In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative procedure that leads to donor cell chimerism and donor specific tolerance. However, most clinical applications of IUHCT have failed because of low levels or even no engraftment of donor cells in immunologically normal fetuses. It is likely that the competition from the host hematopoietic compartment is the primary barrier for successful IUHCT, suggesting that conditioning methods that provide a competitive advantage to donor cell may lead to a higher-level engraftment following IUHCT. This study aimed to research whether maternal administration of low-dose total body irradiation (TBI) or busulfan (BU) before in utero hematopoietic cell transplantation (IUHCT) may result in an increase of donor cell chimerism in postnatal bone marrow transplantation in a congenic murine model. We first test the birth and mortality rate after maternal administration of low-dose TBI (0, 2 or 4Gy) or BU (5, 10, 15 or 20mg/kg) before IUHCT in B6 mice. The mice received 2Gy TBI plus IUHCT showed a significantly decreased birth (23.3%) and 100% 3-day mortality rate. The mice received 10mg/kg BU plus IUHCT showed similar birth and 3-day mortality rate (58.6% and 0%) compared to IUHCT alone (61.1% and 4.55%). We then performed maternal administration of one of three BU dosages (5, 10, or 15 mg/kg) 24 hours before intrauterine transplantation of 2.5 × 105 B6GFP Sca-1+ bone marrow cells (BMCs) or 2.5 × 106 B6GFP BMCs on gestational day 14 (E14). GFP chimerism in PBMC, RBC, and PLT of mice at 4 weeks of age was all enhanced significantly with an increase in the dose of BU. Moreover, GFP chimerism of PBMC from the B6GFP BMCs group was significantly higher than that of the B6GFP Sca-1 + BMCs group (22.56% vs. 7.20%, P = 0.018). Lastly, the pregnant mice were treated with 10 mg/kg of BU at E13, E14, or E15, followed by intrauterine transplantation of 2.5 × 106 B6GFP BMCs 24 h later. Except for the short-term level of chimerism in PBMC, which showed no significant difference among these three groups, the results indicated that both short-term (4 weeks of age) and long-term (14 weeks of age) engraftment in PBMC, RBC, and PLT of group E16 were higher than those of groups E14 and E15. We also discovered that the engraftment was stable, multilineage, and grew with time. In conclusion, maternal administration of BU, but not TBI plus IUHCT, could significantly enhanced engraftment in a congenic murine model.