Prevention of autoimmune diabetes and induction of islet transplantation tolerance in nonobese diabetic (NOD) mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI) conditioning of the recipients. The toxicity of radiation and potential for graft-versus-host disease (GVHD) prevents its clinical application. Donor CD8⁺ T cells play a critical role in facilitation of engraftment but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8⁺ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 monoclonal antibody (mAb). The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (ie, tumor necrosis factor α [TNF-α]), high-level production of anti-inflammatory cytokines (ie, interleukin 4 [IL-4] and IL-10), and confining of the donor CD8⁺ T-cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor-specific tolerance and reversal of insulitis. These results demonstrate that donor CD8⁺ T-cell–mediated facilitation of engraftment can be separated from GVHD in nonirradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.