To clarify major histocompatibility complex (MHC) restriction between hematopoietic stem cells (HSCs) and microenvironments, T cell-depleted bone marrow cells (BMCs) were transplanted into MHC-compatible and MHC-incompatible recipients. A significantly large number of spleen colony-forming units (CFU-S) on day 12 were noted in MHC-compatible recipients, while only a small number were observed in MHC-incompatible recipients. There was, however, no significant difference in CFU-S counts on day 8 between the two groups. A large number of CFU-S counts on day 12 were also observed in F1 hybrid recipients, as seen in syngeneic recipients. The decrease in CFU-S counts on day 12 in MHC-incompatible recipients was also observed even after in vivo abrogation of T and NK cells. The difference in CFU-S counts on day 12 became more prominent when HSC-enriched cells were transferred. These results suggest that an MHC restriction exists between pluripotent HSCs (P-HSCs) and spleen microenvironments. Furthermore, experiments using B10.A recombinant strains revealed that H-2D and S loci play a crucial role in the MHC restriction. The experiments of serial transplantation suggest that the differentiation and proliferation of P-HSCs are inhibited in MHC-incompatible microenvironments. It is therefore likely that the MHC-compatible microenvironment is essential to the differentiation and proliferation of P-HSCs.