Although Tregs are vital for peripheral tolerance, adoptive transfer of Tregs is insufficient in inducing long-term allograft survival in lymphoreplete hosts without preconditioning or adjunct immunosuppression. We hypothesize that reducing donor-reactive T cells may create a therapeutic window for Tregs to induce allograft tolerance without additional immunosuppression. We preconditioned recipient mice using donor-specific transfusion followed by cyclophosphamide treatment to selectively kill proliferating donor-reactive cells. This approach deleted 70-90% donor-reactive T cells, but failed to prolong islet allograft survival. Addition of Tregs with direct donor reactivity prior to transplantation led to long-term survival of BALB/c islets in 70% of C57BL/6 recipients. Equal numbers of polyclonal Tregs significantly prolonged graft survival, but 90% of the recipients rejected their grafts by 100 days. Increasing the numbers of polyclonal Tregs was able to confer long-term graft protection. Similar strategy of combining donor-reactive T cell deletion and polyclonal Tregs did not protect C3H islets in autoimmune NOD recipients. Further addition of Tregs specific for islet autoantigen was required to prolong graft survival in NOD mice. Together, these results demonstrate a clinically viable approach to harness the tolerogenic property of Tregs for inducing long-term allograft acceptance without additional immunosuppression.