The immunosuppressive drugs, cyclosporine A (CsA), tacrolimus, or sirolimus, were analyzed as single agents and in combination with anti-CD40L monoclonal antibody (mAb) for their effects on alloengraftment in mice conditioned with minimal total body irradiation (TBI). Whereas anti-CD40L mAb facilitated chimerism, neither sirolimus nor CsA resulted in substantial alloengraftment. However, sirolimus was synergistic with anti-CD40L mAb for inducing donor chimerism. Contrary to expectations, CsA, a T-cell receptor (TCR) signaling inhibitor, did not abrogate anti-CD40L mAb-facilitated engraftment but rather increased engraftment in anti-CD40L mAb-treated mice. Although tacrolimus alone or with anti-CD40L mAb resulted in similar levels of donor chimerism, donor T-cell reconstitution was very low in tacrolimus-treated mice. At 1 week after transplantation, CsA decreased thymic numbers more profoundly than sirolimus or tacrolimus in anti-CD40L mAbtreated recipients. In contrast, only sirolimus resulted in a decrease in host splenic T-cell numbers in anti-CD40L mAbtreated recipients. Importantly, sirolimus and anti-CD40L mAb induced profound donor tolerance with 100% acceptance of donor skin grafts placed early after bone marrow transplantation (BMT). In contrast, anti-CD40L mAb alone or in combination with CsA resulted in 12% or less donor skin graft acceptance early (1 month) and 60% or less later (3 months) after BMT. These data have clinical relevance and indicate that immunosuppressive pharmacologic agents enhance anti-CD40L mAb-facilitated alloengraftment and tolerance induction under nonmyeloablative conditioning.