We recently developed a murine protocol for the induction of allogeneic mixed chimerism and tolerance employing nonmyeloablative total body irradiation (TBI), standard-dose bone marrow transplantation (BMT), and costimulation blockade (cobl) with an anti-CD154 monoclonal antibody (mAb) plus CTLA4Ig. We now evaluated whether a short course (1 month) of immunosuppressive drugs, which would be ethically required in the clinical setting of organ transplantation to prevent graft loss in case tolerance is not achieved, interferes with tolerance induced with this regimen. Our results show that calcineurin inhibitors (cyclosporin A [CyA] or tacrolimus [FK]) inhibit development of long-term chimerism and abrogate tolerance induction in this model. Rapamycin (rapa), methylprednisolone (MP), FTY720, and mycophenolate mofetil (MMF), in contrast, have no negative effect on chimerism or tolerance development. Peripheral deletion of donor-reactive T cells, which usually occurs in the weeks following BMT in this model, is blocked by CyA and FK, but not by the other drugs tested. Furthermore, we found that the additional use of compatible immunosuppressive drugs (rapa plus MMF plus MP) allows the dose of TBI to be reduced, so that mixed chimerism and donor skin-graft acceptance can be achieved with 1 Gy using clinically feasible cell numbers. Thus, this protocol of BMT with costimulation blockade can be safely combined with a clinically tested immunosuppressive regimen to permit success with a lower dose of irradiation. These results should facilitate clinical application of this tolerance strategy.