Once the Act 3 Boss has been defeated with all characters except the Watcher at least once in a normal run, three keys will begin to appear in subsequent normal runs. If all three keys are collected in the run, the run will not end after the Act 3 boss; instead, the player will enter Act 4.
The Ruby Key is obtained by taking the Recall action at Rest Sites. The key may be obtained at any Rest Site along the run.
The Sapphire Key is obtained from a Treasure Room chest (including chests found in ? Rooms). It appears linked with a Relic, such that only the key or the relic may be taken. If the relic is taken, the key will keep appearing in subsequent chests until it is taken.
If you have the relic Matryoshka，you'll be able to take one of the relics freely, since the additional relic won't be linked with the Sapphire Key.