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Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development in the context of normal immune ontogeny. Methods Bone marrow (BM) cells (10x106) from C57/BL6 (B6, H2kb) mice were injected intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). E14 B6 fetuses injected with GFP B6 BM were used as congeneic controls. At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. T cell alloreactivity was assessed by in vitro and in vivo MLR. Results Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of single positive (SP) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor TCR gamma/delta cells in DN cells which do not require positive selection based on MHC recognition are relatively increased compared to the host. Moreover, compared with the naive mice and congeneic chimeric mice, the donor BM-derived thymocytes in the allogeneic chimeric mice show increased proportion of DN3 and decreased proportion of DN4, but increased TCRβ+ proportions in both DN3 and DN4 cells, indicating that donor BM-derived thymocyte development is impeded during DN to DP transition, resulting from a MHC-restriction associated mechanism. In addition, in allogeneic chimeric mice, both host and donor BM-derived T cells are tolerant to allogeneic antigens in in vivo and in vitro MLR. Conclusion Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. Taken together, although donor BM-derived T cells undergo differential thymic development, permanent host-donor two-way tolerance could be achieved in the allogeneic IUHCT mouse model. These findings add to our understanding of the requirements for tolerance induction after IUHCT and have important clinical implications in choosing an optimal donor for IUHCT. Disclosures No relevant conflicts of interest to declare.