We previously demonstrated that even a low dose of bone marrow cells (BMCs) established donor-specific tolerance if mixed with splenocytes (SPLCs). In this study, T-cell subsets CD4 (CD4SP) and CD8 (CD8SP) of donor SPLCs were investigated for their contribution to the enhancement of BMC engraftment leading to donor-specific tolerance in sublethally irradiated mice. Sublethally irradiated C57BL/6 recipient mice were intravenously injected BMCs mixing with CD4SP or CD8SP harvested from BALB/c donor mice. The degree of chimerism in the peripheral blood lymphocytes (PBLs) and in the SPLCs was analyzed using FACS, mixed lymphocyte reaction, and skin graft transplantation 3 months after injection. Recipients injected with 3 × 10 6 donor BMCs admixed with 10 × 10 6 donor CD8SP established chimerism. However, recipients injected with the same dose of BMCs admixed with 5 × 10 6 CD4SP, 10 × 10 6 CD4SP, and 5 × 10 6 CD8SP did not established chimerism. CD8SP contained 44% of Ly6A/E (Stem Cell Antigen-1 (Sca-1))–positive cells based on FACS analysis, whereas only 6% of CD4SP were positive for Ly6A/E. MLR supernates of donor SPLCs chimeric mice using admixture with CD8SP dominated by Th2 cytokines. In contrast, mixting with MLR supernates from failed chimera showed dominant Th1 cytokines. CD8SP seems to make a major contribution to enhance BMC engraftment and induce donor-specific tolerance. Ly6A/E (Sca-1)–positive cells need to be further investigated for their contribution to the establishment of chimerism.