How to Re-enter Vault 101 in Fallout 3 (Xbox 360) — It's Possible Later in the Story

You can return to Vault 101 later in the main questline, contrary to popular belief.

The Quest: "Trouble on the Homefront"

1. Progress through the main story until after "The Waters of Life" (Project Purity activation).
2. A distress signal from Amata will trigger the quest "Trouble on the Homefront" on your Pip-Boy radio.
3. Fast travel to Vault 101 marker; the door will now be accessible.
4. Resolve the internal conflict (overseer, rebels, etc.).

After completing this quest, the Vault seals permanently again. You cannot enter a third time.