Preactivated CD4⁺CD25⁺ regulatory T cells durably prevent semiallogeneic bone marrow rejection by total splenocytes. 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) and coinjected with 10⁵ B6 splenocytes and regulatory T cells preactivated with B6D2F1 APCs in vitro (regulatory-to-effector T-cell ratio as indicated). Bone marrow was analyzed 2 weeks later (A-B; combined data from 4 independent experiments), and PBMCs were analyzed at indicated time points (C; representative individual mice). (A) FACS analysis of bone marrow. (B-C) Relative percentage of semiallogeneic cells (see “Materials and methods”). ^***P < .001 (Student t test). Horizontal bars in panel B indicate the mean.