It's uncertain, but one possibility is that
O'Brien believes that Winston must already know what is in Room 101: one's worst fears made reality.
‘What is in Room 101?’
The expression on O’Brien’s face did not
change. He answered drily: ‘You know what is in Room 101, Winston.
Everyone knows what is in Room 101.’
—1984, p. 328
For a moment he was alone, then the door opened and O’Brien came in.
‘You asked me once,’ said O’Brien, ‘what was in Room
101. I told you that you knew the answer already. Everyone knows it. The thing that is in Room 101 is the worst thing
in the world.’
—1984, p. 357
The implication is that no one needs to be told what is in Room 101, that they will know what it contains as soon as they know of it. Indeed, this makes sense, since it contains one's worst fear; without information, a person's mind imagines the most horrible things, which is precisely what Room 101 contains.
Obviously, he initially does not know.
Only once, when two Party members, both women, were pressed close
together on the bench, he overheard amid the din of voices a few
hurriedly-whispered words; and in particular a reference to something
called ‘room one-oh-one’, which he did not understand.
—1984, p. 298
It is likely only once he is told that he knows what is in Room 101, and thus begins to imagine the worst horrors conceivable to him, that he then knows what is in Room 101.
The other prisoners probably suspect that he has gone through this process.