Despite its wide use as a marker for hematopoietic stem cells (HSCs), the function of stem cell antigen–1 (Sca-1) (also known as lymphocyte activation protein–6A [Ly-6A]) in hematopoiesis remains poorly defined. We have previously established that Sca-1^−/−T cells develop normally, although they are hyperresponsive to antigen. Here, we report detailed analysis of hematopoiesis in Sca-1–deficient animals. The differentiation potential of Sca-1–null bone marrow was determined from examination of the most mature precursors (culture colony-forming units [CFU-Cs]) to less committed progenitors (spleen CFUs [CFU-Ss]) to long-term repopulating HSCs. Sca-1–null mice are mildly thrombocytopenic with a concomitant decrease in megakaryocytes and their precursors. Bone marrow cells derived fromSca-1^−/− mice also have decreased multipotential granulocyte, erythroid, macrophage, and megakaryocyte CFU (GEMM-CFU) and CFU-S progenitor activity. Competitive repopulation assays demonstrated that Sca-1^−/−HSCs are at a competitive disadvantage compared with wild-type HSCs. To further analyze the potential of Sca-1^−/−HSCs, serial transplantations were performed. While secondary repopulations using wild-type bone marrow completely repopulatedSca-1^−/−mice, Sca-1^−/−bone marrow failed to rescue one third of lethally irradiated wild-type mice receiving secondary bone marrow transplants from irradiation-induced anemia and contributed poorly to the surviving transplant recipients. These data strongly suggest that Sca-1 is required for regulating HSC self-renewal and the development of committed progenitor cells, megakaryocytes, and platelets. Thus, our studies conclusively demonstrate that Sca-1, in addition to being a marker of HSCs, regulates the developmental program of HSCs and specific progenitor populations.