While the majority of purified pluripotential hematopoietic stem cells (PHSC) express c-Kit, the receptor for steel factor, we have phenotypically and functionally separated a distinct class of PHSC that does not express c-Kit. In contrast to c-Kit-postive (c-Kit^pos) PHSC, the c-Kit-negative (c-Kit^neg) PHSC do not proliferate in response to multiple hematopoietic growth factors in vitro and do not radioprotect or form macroscopic spleen colonies (CFU-s) when transplanted into lethally irradiated recipients. However, the c-Kit^neg PHSC show delayed or slow reconstitution kinetics when cotransplanted with radioprotective bone marrow cells. c-Kit^neg PHSCs cells can give rise to c-Kit^pos cells with CFU-s activity, radioprotective activity, and PHSC activity. Thus, constituitive hematopoiesis is maintained by c-Kit^pos PHSCS cells that are recruited from a more primitive quiescent c-Kit^neg PHSC population, which represents a critical developmental stage in definitive hematopoiesis.