Introduction: Traditional techniques for bone marrow transplantation (BMT) rely on the production of biologic “space” in the recipient bone marrow compartment with myeloablative conditioning. A major disadvantage to myeloablative conditioning is a transient period of myelosuppression prior to bone marrow (BM) reconstitution, during which the BMT recipient is susceptible to infection. The purpose of this study is to describe a reliable, well tolerated, and labor non-intensive technique for the production of stable mixed hematopoietic chimerism without myeloablative conditioning. Methods: SJL mice served as the universal BM donors. SJLB6F1 (F1) or C57BL/6 (B6) mice served as the recipient strains. Recipient mice received sublethal total body irradiation (TBI) on day -1, 200 mg/kg intraperitoneal (i.p.) Cyclophosphamide on days –3 and +3, and donor BM on day 0. TBI doses of 200 to 500 cGy and cell doses from 10 × 106 to 40 × 106 were evaluated. Hematopoietic chimerism was determined with flow cytometry at 21 and 60 days post-engraftment. Results: 28/28 (100%) mice in the haploidentical strain combination (SJL into F1) developed 1%–19% chimerism by 21 days. Chimerism in all of these mice increased to 15%–61% by 60 days and remained stable in that range. A BM cell dose of 20 × 106 and a minimum TBI dose of 200 cGy was adequate. In the fully allogeneic strain combination (SJL into B6), 14/22(64%) mice developed 1%–15% chimerism by 21 days. This increased to 3%–56% by 60 days and subsequently remained stable in that range. A donor BM cell dose of 20 × 106 was effective. However, a minimum TBI dose of 500 cGy was necessary for the allogeneic strain combination. TBI of less than 500 cGy was inadequate for stable mixed chimerism production. Conclusions: Mixed chimerism can be effectively produced in a haploidentical and fully allogeneic strain combination using nonmyeloablative conditioning. A minimum TBI dose of 500 cGy in addition to a well-tolerated dose of i.p. Cyclophosphamide is necessary for engraftment within the allogeneic strain combination. The TBI dose can be effectively dropped to 200 cGy for engraftment within a haploidentical (parent into F1) model.