Background. Tolerance induction is an important goal in the field of organ transplantation. We have sequentially modified our conditioning regimen for induction of donor-specific tolerance in recipients of major histocompatibility complex-mismatched combined kidney and bone marrow transplantation (CKBMT). Methods. From December 2011 to May 2017, 8 major histocompatibility complex-mismatched patients received CKBMT. The initial conditioning regimen (protocol 1) consisted of cyclophosphamide (CP), rituximab, rabbit antithymocyte globulin, and thymic irradiation. Tacrolimus and steroids were used for the maintenance of immunosuppression (IS). Results. This regimen was complicated by transient acute kidney injury, which has been the major clinical feature of engraftment syndrome and side effects of CP, although one of 2 subjects successfully discontinued his IS for 14 months. The conditioning regimen was modified by reducing the CP dose and adding fludarabine (protocol 2). The final modification was reducing the fludarabine and rabbit antithymocyte globulin doses (protocol 3). Mixed chimerism, detected by the short tandem repeat method, was achieved transiently in all subjects for 3-20 weeks. Among the 3 subjects treated with protocol 2, IS was successfully discontinued for >35 months in one subject, but the other 2 subjects suffered from severe BK virus-associated nephritis. All 3 subjects treated with protocol 3 tolerated the protocol well and have successfully discontinued IS for >4-41 months. Interestingly, de novo donor-specific antibody was not detected in any subject during all the follow-up periods. Conclusions. Our clinical trial has shown that long-term renal allograft survival without maintenance IS can be achieved by induction of mixed chimerism following CKBMT.