Objective. To investigate the possibility of flk-1＋Sca-1- bone marrow-derived mesenchymal stem cells (bMSCs) to induce stable mixed chimerism and donor-specific graft tolerance. Methods. Allogeneic flk-1＋Sca-1- bMSCs and syngeneic bone marrow (BM) cells were cotransplanted into lethally irradiated (8.5 Gy) recipient mice. FACS was used to analyzethe chimerism 150 days later. Donor-type skin transplantation was performed to observe donorspecific immunotolerance in recipient mice. Mixed lymphocyte reaction (MLR) and mitogen proliferative assays were performed to evaluate proliferative response of splenocytes from recipient mice. Results. More than 5% donor-derived CD3＋ cells were detected in splenocytes of recipientmice. Long-term survival of donor-type skin grafts was observed. MLR and mitogen proliferative assays showed that recipient mice had low immunoresponse to donor cells butretained normal ConA-induced proliferative response compared with normal mice. Conclusion. Our results show for the first time that induction of stable mixed hematopoietic chimerism can be achieved with allogeneic flk-1＋Sca-1-　bMSC transplantation, which leadsto permanent donor-specific immunotolerance in allogeneic host and results in long-term allogeneic skin graft acceptance.