Boos first appeared in Super Mario Bros. 3. These ghosts froze and covered their face if Mario looked at them, but followed him if he did not. They can only be destroyed if light shines on them (in the Super Mario Galaxy series), if Mario hits them with a Star powerup, or with a Spiny Shell in New Super Mario Bros. for the DS.
In Super Smash Flash 2
Boos were initially confirmed to appear as enemies in the Mushroom Kingdom level for the game's Adventure mode. Any further information in unknown and whether it will appear is no longer confirmed.
Another Boo appears as a background character in the Waiting Room's Halloween theme, one of multiple holiday-inspired versions of the stage. It can be seen floating above the left couch in the background.