Type 1 diabetic NOD mice have defects in both thymic negative selection and peripheral regulation of autoreactive T cells, and induction of mixed chimerism can effectively reverse these defects. Our recent studies suggest that MHC-mismatched mixed chimerism mediates negative selection of autoreactive thymocytes in wild-type NOD and TCR-transgenic NOD. Rag1+/+. BDC2. 5 mice. However, it remains unknown how mismatched IA b MHC class II can mediate deletion of autoreactive T cells positively selected by IA g7. In the present study, we directly tested the hypothesis that mismatched MHC class II in mixed chimeras mediates deletion of cross-reactive autoreactive thymocytes. We first identify that transgenic BDC2. 5 T cells from NOD. Rag1+/+. BDC2. 5 but not NOD. Rag1−/−. BDC2. 5 mice possess cross-reactive TCRs with endogenous TCRα-chains; MHC-mismatched H-2 b but not matched H-2 g7 mixed chimerism mediates thymic deletion of the cross-reactive transgenic T cells in NOD. Rag1+/+. BDC2. 5 mice. Second, by transplanting T cell–depleted (TCD) bone marrow (BM) cells from NOD. Rag1+/+. BDC2. 5 or NOD. Rag1−/−. BDC2. 5 mice into lethally irradiated MHC-mismatched H-2 b C57BL/6 or MHC-matched congenic B6. H-2 g7 recipients, we demonstrate that NOD. Rag1+/+. BDC2. 5 BM-derived cross-reactive transgenic T cells, but not NOD. Rag1−/−. BDC2. 5 BM-derived non–cross-reactive transgenic T cells, can be positively selected in MHC-mismatched H-2 b thymus. Third, by cotransplanting NOD. Rag1+/+. BDC2. 5 TCD BM cells with BM cells from MHC-mismatched T cell–deficient C57BL/6 mice into lethally irradiated MHC-matched B6. H-2 g7 recipients, we demonstrate that thymic deletion of the cross-reactive transgenic T cells is dependent on MHC-mismatched donor BM-derived APCs but not on donor BM-derived T cells. Taken together, our studies indicate that MHC-mismatched mixed chimerism can mediate thymic deletion of cross-reactive autoreactive T cells that express more than one TCR.