Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin−CD34+CD38− cord blood and bone marrow and express CD90. We demonstrate that the Lin−CD34+CD38− fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA−, CD90−CD45RA−, and CD90−CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin−CD34+CD38−CD90+CD45RA− cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin−CD34+CD38−CD90−CD45RA− cord blood.