We reported stable mixed chimerism and specific tolerance to a fully allogeneic graft after a minimally myelosuppressive regimen including costimulation blockade (CB), donor bone marrow cells (BMC), and busulfan (Bu), a chemotherapeutic conditioning agent that makes niches for engraftment of BMC. For clinical application, the strategy may have the limitation of the number of donor BMC when a deceased donor offers transplants to multiple recipients. Herein, we examined whether donor splenocytes can serve as an alternative source to induce mixed chimerism and tolerance. When a C57BL/6 (H-2b) recipient was treated with CB (CTLA4-Ig and anti-CD154 mAb, on days 0, 2, 4, 6) and donor BALB/c (H-2d) BMC (2 x 10(7) cells on day 0) in the absence of Bu, survival of BALB/c skin graft was remarkably prolonged but not indefinite (median survival time [MST]: 138 days). The recipients never showed durable chimerism. When the recipient was treated with CB and donor splenocytes ([DST] 2 x 10(7) cells on day 0), survival was not indefinite either (MST: 114 days). When the dose of DST was increased to 2 x 10(8) cells, survival was further prolonged; two of six recipients had indefinite survival (MST: 132 days). Moreover, one recipient showed a low level of chimerism. When treated with CB, donor DST (2 x 10(7) cells on day 0) and Bu (20 mg/kg, day -1), six of seven recipients showed a stable, high level of chimerism and enjoyed tolerance of skin allografts. DST combined with CB and Bu may be an alternative source of hematopoietic stem cells to induce mixed chimerism and transplantation tolerance in our model.