CD4⁺CD25⁺ regulatory T cells preactivated in vitro induce CD4⁺ and CD8⁺ T-cell tolerance to semiallogeneic bone marrow. B6 mice were lethally irradiated and grafted with a 1:1 mixture of B6 (H-2^b) and B6D2F1 (H-2^bd) bone marrow (5 × 10⁶ cells each). Two weeks later equivalent percentages of syngeneic and semiallogeneic bone marrow were observed in bone marrow (A). Irradiated mice were coinjected with the 2 types of bone marrow and 3 × 10⁵ CD4⁺ (panel B, left column; panel C) or CD8⁺ T cells (panel B, right column; panel D) with (panel B, bottom row) or without (panel B, top row) CD4⁺CD25⁺ regulatory T cells from B6 mice in vitro preactivated with B6D2F1 APCs (panel B; regulatory-to-effector T-cell ratio = 10; panels C-D as indicated). Mice were analyzed 2 weeks later. (Panel A, top; panel B) FACS analysis of bone marrow, (panels C-D) relative percentage (see “Materials and methods”) of semiallogeneic bone marrow. ▪ indicates individual mice from a representative experiment of 4 performed. Error bars in panel A indicate SEM.