Thanks naz, only 16 for normal luck is an insanely low answer, I legit thought it would need to be like 30+. And yeah both of these would be a pain luck and skill wise It's possibility doable. The first idea may need some theory testing just in case like something about the run makes it undoable like not having assess to a needed item.

Originally posted by Slushea:

The first idea may need some theory testing just in case like something about the run makes it undoable like not having assess to a needed item.

According to the wiki both the Glowing Hourglass and the shovel are available by default (no unlock requirement), and I tested if Depths 2 was skippable and if the hourglass worked like that on a fresh save (using the debug console to give myself the items). And they do.
But actually getting both items like that would be crazy RNG, indeed.


Edit:
I just imagined a crazier combo... But it relies on one thing I'm not sure of.
When you unlock an item, can you generate that item during the same run or do you have to wait for the next?
I genuinely do not know the answer to that question. Requires testing - typically, unlock an item during a run, once you do use a D6 with infinite charges to deplete its item pool until you see it or deplete it. I'll do that later once I have some time.

Assuming you can generate it in the same run, the absolute minimum would become 1 run.
On each of the 6 floors, enter the shop (this assumes you never get an XL floor). Entering the 6th shop unlocks the Schoolbag item (hold two active items). Generate it somehow (a 5-pips dice room on the 6th floor would reset it and get you a new shop, for example).
Find both the R Key and the Glowing Hourglass (one can be found before getting the Schoolbag, makes it easier).
Now use the R Key. You're back in the starting room of Basement 1, and you're holding the Glowing Hourglass. Use it, and you're at Home. And here you have more items to kill the Beast than in the previous 2-runs scenario.

Edit 2:
Just tested it. Unlocked the schoolbag during a run (on another save), and I couldn't get it during the same run with infinite rerolls (unless I somehow missed it). Got it in the next run though.
So the minimum seems to be 2 runs. Oh well. At least I learned items are unlocked for later runs, not the current one.

Ah Still pretty cool the lowest amount required is 2 with godly rng, 16 with more regular rng. I also now wonder what would also be like the most amount of character unlocks doable in the 16 run. I know lilith and lost are 100% out of the picture as one requires greed mode and the other requires you die. Also Thanks man for the answer.

Originally posted by Slushea:

I also now wonder what would also be like the most amount of character unlocks doable in the 16 run.

Well, let's see how likely each of the 17 characters are...

Isaac: unlocked by default. Duh.

Magdalene: 7+ red hearts.
Possible.

Cain: 55+ coins.
Possible.

Judas: kill Satan.
Possible.
While Satan isn't in the process, it is possible to get a Devil Deal when killing the Heart, which leads to Sheol before It Lives is unlocked.
But even without that, starting at the 11th Heart kill Sheol is always accessible, so the 2nd and 3rd Hush kills could then lead to a Satan one (1st Hush kill forces an ending).

??? (Blue Baby): 10th Heart kill.
Forced.
Unlocked at the same time as Hush.

Eve: don't pick up health for two floors.
Possible.

Samson: complete two floors without taking damage.
Possible.

Azazel: pay health for 3 devil deals in one run.
Possible.

Lazarus: 4+ soul hearts.
Possible.

Eden: first Heart kill.
Forced.

the Lost: die in a sacrifice room with the Missing Poster effect active.
Possible.
While playing as Isaac, after killing the Heart, use a sacrifice room to teleport to the Dark Room and kill the Lamb to unlock the Missing Poster (can technically be done during the very first run, but a Heart kill run makes it more practical for later runs).
Alternatively, get 5 devil deals when killing the heart to reach Sheol and unlock the negative (1st-5th Heart kills runs). You can then reach the Dark Room by getting other devil deals (6th Heart kill run at the earliest).
During a later run, kill yourself in a sacrifice room while holding the poster. You will turn into the Lost, it will be unlocked, and you can continue the run (at least according to the wiki). Worst case, get an extra life item first to make sure the run continues.

Lilith: beat Greed mode as Azazel.
Not possible.

Keeper: 1000 coins in the Greed machine.
Not possible.

Apollyon: first Mega Satan kill.
Possible.
Requires having killed ??? or the Lamb once, and the Lamb has already been covered before. Next run, Mega Satan can be reached. (7th Heart kill run, at the earliest.)

The Forgotten: (it's complicated)
Possible.
The first step is to kill the Lamb, but we already covered that. The unlock run needs to kill Mom then reach the Dark Room, but again, we know how to do it, one way or another (7th Heart kill run, at the earliest, so don't forget to kill Mega Satan while you're there).

Bethany: kill the Heart as Lazarus on hard mode without using your extra life.
Possible.
Once Lazarus is unlocked, this only takes one more run. And Lazarus can be unlocked during the very first run.

Jacob & Esau: kill Mother.
Forced.
Unlocked at the same time as the Ascent path.


Overall, out of the 17 characters:

• 1 is unlocked by default. (Isaac)
  
• 3 are forced. (???, Eden, Jacob & Esau)
  
• 2 aren't possible. (Lilith, Keeper)
  
• 11 are possible. (all the others)

And add one tainted character, depending on whoever you are for the Beast run (Red Key is forced the first time you go to the Home floor).

Edit:
(Now I love this thread for some reason. Please don't delete it.)

So in total, 16 characters are unlockable (13 if we exclude Bethany, lost and forgotten as they are sorta odd). As that's excluding the 2 impossible ones and including the one tainted one you'd get during the final run. I now kinda wondering how many runs for all of the endings to be unlocked. (Assuming you got one of the heart endings each run to prevent RNG from ruining the answer. )