This study describes the purification of primitive human hematopoietic stem cells capable of repopulating immune-deficient mice. Using a two-step strategy involving lineage-depleted cells followed by fluorescence-activated cell sorting, researchers purified a cell population enriched for cells that can repopulate nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These cells, termed SCID-repopulating cells (SRCs), were found exclusively in a fraction expressing high levels of CD34 and no CD38. Limiting dilution analysis showed a frequency of 1 SRC in 617 CD34+ CD38- cells. The highly purified SRCs were capable of extensive proliferation and differentiation in NOD/SCID mice, with mice transplanted with 1 SRC producing approximately 400,000 progeny six weeks after the transplant. Flow cytometric analysis of the marrow of engrafted mice showed both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+ CD38- cells. These highly purified fractions are useful for identifying the cellular and molecular mechanisms regulating primitive human hematopoietic cells. The ability to detect and purify primitive cells also provides a means to develop conditions for maintaining and expanding these cells during in vitro culture. The hematopoietic system in mammals consists of a heterogeneous population of cells with varying functions, ranging from mature cells with limited proliferative potential to pluripotent stem cells with extensive proliferative, differentiative, and self-renewal capacities. Stem cells play a key role in maintaining the hematopoietic system and are being targeted for clinical applications such as gene therapy, ex vivo expansion, and cord blood transplantation. However, progress in characterizing human stem cells has been hindered by their rarity, lack of a distinctive phenotype, and absence of a functional repopulation assay. Research has relied on surrogate in vitro assays, including clonogenic assays and long-term culture-initiating cells (LTC-ICs). Recent studies suggest that a small subpopulation of LTC-ICs has more extensive proliferative capacity and can be maintained in extended LTC for up to 100 days. However, the relationship between LTC-ICs and pluripotent stem cells remains unclear. The only conclusive assay for stem cells is their ability to reconstitute the entire hematopoietic system after transplantation. Based on in vitro assays and human allogeneic transplantation, the most primitive hematopoietic cells are enriched in the CD34+ fraction. CD34+ cells are heterogeneous and include committed progenitors. By assessing coexpression of other cell surface markers, it is possible to obtain cell fractions more enriched for primitive cells. CD38 expression is correlated with increased differentiation; only 1–10% of CD34+ cells do not express CD38. Both the CDThis study describes the purification of primitive human hematopoietic stem cells capable of repopulating immune-deficient mice. Using a two-step strategy involving lineage-depleted cells followed by fluorescence-activated cell sorting, researchers purified a cell population enriched for cells that can repopulate nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These cells, termed SCID-repopulating cells (SRCs), were found exclusively in a fraction expressing high levels of CD34 and no CD38. Limiting dilution analysis showed a frequency of 1 SRC in 617 CD34+ CD38- cells. The highly purified SRCs were capable of extensive proliferation and differentiation in NOD/SCID mice, with mice transplanted with 1 SRC producing approximately 400,000 progeny six weeks after the transplant. Flow cytometric analysis of the marrow of engrafted mice showed both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+ CD38- cells. These highly purified fractions are useful for identifying the cellular and molecular mechanisms regulating primitive human hematopoietic cells. The ability to detect and purify primitive cells also provides a means to develop conditions for maintaining and expanding these cells during in vitro culture. The hematopoietic system in mammals consists of a heterogeneous population of cells with varying functions, ranging from mature cells with limited proliferative potential to pluripotent stem cells with extensive proliferative, differentiative, and self-renewal capacities. Stem cells play a key role in maintaining the hematopoietic system and are being targeted for clinical applications such as gene therapy, ex vivo expansion, and cord blood transplantation. However, progress in characterizing human stem cells has been hindered by their rarity, lack of a distinctive phenotype, and absence of a functional repopulation assay. Research has relied on surrogate in vitro assays, including clonogenic assays and long-term culture-initiating cells (LTC-ICs). Recent studies suggest that a small subpopulation of LTC-ICs has more extensive proliferative capacity and can be maintained in extended LTC for up to 100 days. However, the relationship between LTC-ICs and pluripotent stem cells remains unclear. The only conclusive assay for stem cells is their ability to reconstitute the entire hematopoietic system after transplantation. Based on in vitro assays and human allogeneic transplantation, the most primitive hematopoietic cells are enriched in the CD34+ fraction. CD34+ cells are heterogeneous and include committed progenitors. By assessing coexpression of other cell surface markers, it is possible to obtain cell fractions more enriched for primitive cells. CD38 expression is correlated with increased differentiation; only 1–10% of CD34+ cells do not express CD38. Both the CD