In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism and donor-specific tolerance without cytoreductive conditioning or immunosuppression. The primary limitation to the clinical application of IUHSCTx has been minimal donor cell engraftment, well below therapeutic levels for most target diseases. Donor lymphocyte infusion (DLI) has been used in postnatal circumstances of mixed chimerism as targeted immunotherapy to achieve a graft-versus-hematopoietic effect and to increase levels of donor cell engraftment. In this report we demonstrate in the murine model that a combined approach of IUHSCTx followed by postnatal DLI can convert low-level, mixed hematopoietic chimerism to complete donor chimerism across full major histocompatibility complex barriers with minimal risk for graft-versus-host disease (GVHD). Time-dated embryonic day 14 (E14) to E15 Balb/c (H-2K(d), CD45.2) fetuses underwent intraperitoneal injection of 5 x 10(6) T-cell-depleted B6 (H-2K(b), CD45.2) bone marrow cells. Chimeric recipients then received transplants at either 4 or 8 weeks of age with 1 of 3 doses (5, 15, or 30 x 10(6) cells) of donor congenic splenocytes (B6-Ly5.2/Cr, H-2K(b), CD45.1). The response to DLI was dose dependent, with conversion to complete donor peripheral blood chimerism in 100% of animals that received high-dose (30 x 10(6) cells) DLI. Only 1 of 56 animals receiving this dose succumbed to GVHD. This study directly supports the potential therapeutic strategy of prenatal tolerance induction to facilitate nontoxic postnatal cellular therapy and organ transplantation, and it has broad implications for the potential treatment of prenatally diagnosed genetic disorders.