Bim is required for induction of mixed chimerism. BM3.3 splenocytes were stimulated in vitro with CD8-depleted B6 (allo) or CBA (syn) splenocytes. The expression of different Bcl-2 factors in transgenic alloreactive BM3.3 CD8 T cells was monitored by FACS. (A) In comparison with allostimulated cells without additional pharmacological treatment, after 4 days of MLR, cells exposed to MR1 expressed higher levels of Bcl-2 and Mcl-1 and lower levels of Bcl-xL. The expression of these antiapoptotic factors was not influenced by an additional treatment with CsA. Percentages of mean fluorescence intensity (MFI) values in comparison with allostimulated cells without pharmacological treatment are shown. (B) Allostimulation induced a transient upregulation of Bim, with a peak after 2 days of culture. CsA inhibited the initial upregulation of Bim, and MR1 prevented its downregulation in the late activation phase. (C) As a result, after 4 days the level of Bim was low in cells stimulated in the presence of CsA and high with MR1. Statistical comparison with syn: *P < .01. (D) The relevance of these processes on CD8 T-cell viability after polyclonal stimulation was assessed culturing WT and Bim^−/− splenocytes in the presence of anti-CD3 with anti-CD28 antibodies or without anti-CD28 and MR1. After 4 days, absence of costimulation reduced the viability of WT CD8 T cells, but the same phenomenon was not observed using Bim^−/− cells, suggesting that the downregulation of Bim (C) was important for the viability of activated T cells. **P < .01. (E) In vivo, a standard conditioning protocol (3 Gy TBI, MR1, 25 × 10⁶ CBA BM cells) induced mixed chimerism in all WT B6 mice, but was not successful in the majority of Bim^−/− mice, as shown by the levels of chimerism 15 weeks after BMT and (F) by the rejection of CBA skin grafts. Similar results were obtained if ABT-737 was added to the same conditioning protocol (E-F). Statistical comparison WT vs Bim^−/−: *P < .05; ***P < .001; N = 6-7 per group.