The results of previous studies have shown that the development of hematopoiesis during fetal life can occur in the absence of Steel factor (SF ) signaling. On the other hand, impairment of this mechanism can severely compromise the ability of cells from adult bone marrow to regenerate hematopoiesis on their transplantation into myeloablated recipients. This apparent paradox could result from changes during ontogeny in the responsiveness of hematopoietic stem cells to regulators that may substitute for SF as well as from differences in the availability of such factors during embryogenesis and in the myeloablated adult. To investigate these possibilities, we studied the effect of W⁴¹ and W⁴² mutations on the numbers, phenotype, and posttransplant self-renewal behavior of primitive hematopoietic cells present in the fetal liver (FL) of 14.5-day-old mouse embryos. In W⁴¹/W⁴¹ FL, day-12 spleen colony-forming units and long-term culture-initiating cells appeared both quantitatively and qualitatively similar to their counterparts in the FL of +/+ embryos. W⁴¹/W⁴¹ FL also contained near normal numbers (≈50% of controls) of transplantable lymphomyeloid stem cells with competitive reconstituting ability in myeloablated adult +/+ recipients (as assessed for up to at least 16 weeks posttransplant). Moreover, both the original phenotype of these W⁴¹/W⁴¹ competitive repopulating units (CRUs) and their clonal posttransplant output of mature progeny were normal. Similarly, when myeloablated adult +/+ mice were cotransplanted with 5 × 10⁴ +/+ FL cells and a sevenfold to 70-fold excess of W⁴¹/W⁴¹ FL CRUs, the contribution of the +/+ FL CRUs to the circulating white blood cell count present 5 weeks later was markedly reduced as compared with that of mice that received only +/+ FL cells. However, over the next 3 months, the proportion of mature white blood cells that were derived from +/+ precursors increased significantly (P < .002) in all groups (to ≥30%), indicating that the ability to sustain hematopoiesis beyond 5 weeks is more SF-dependent than the ability to initially reconstitute both lymphoid and myeloid compartments. Cells from individual FL of W⁴²/+ matings also showed an initial ability (at 7 to 8 weeks posttransplant) to competitively repopulate both lymphoid and myeloid compartments of myeloablated +/+ adult recipients. However, in contrast to recipients of normal or W⁴¹/W⁴¹ FL cells, the repopulation obtained with the W⁴² mutant stem cells was transient. Secondary transplants confirmed the inability of the W⁴² mutant cells to regenerate or even maintain a population of transplantable stem cells. Taken together with previous results from studies of CRUs in adult W mice, these findings support the concept of changes in the way hematopoietic stem cells at different stages of development respond to the stimulatory conditions evoked in the myeloablated recipient. In addition, they provide the first definitive evidence that SF is a limiting physiological regulator of sustained hematopoietic stem cell self-renewal in vivo.