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Identification of phenotypes of human hematopoietic cells that display long-term mature cell outputs in vitro and repopulating capability in immunodeficient mice has been important to anticipating the therapeutic potential of fresh harvests of bone marrow or cord blood before or after their physical or genetic manipulation. Characterizing additional properties of these cells and elucidating the mechanisms that regulate their ability to sustain mature blood cell production has thus remained a major focus of interest. Previous studies have shown that fetal and adult human cells with long-term blood cell output potential are highly enriched in their respective GPI80+ and CD49f+ subsets of a developmentally preserved CD45+CD34+CD38-CD45RA-CD90+ population. The so-called “GPI80” hematopoietic cells found in first trimester human fetal liver are of particular interest because of their very high regenerative capability compared to their adult or even neonatal (cord blood) “CD49f” counterparts. However, in vitro conditions that enable the extensive innate self-renewal capability of either of these cell types to be maintained have remained elusive. The goals of this project were first to test the hypothesis that retention of the high regenerative activity of the GPI80+ cells in vitro would be optimized under different conditions than those that regulate their viability or mitogenesis; and second, that the GPI80+ cells of interest would co-express CD49f. The results show first, that the defining long-term cell output function of human hematopoietic stem cells (HSCs) in fetal liver is best maintained in vitro by FLT3L alone despite its poor ability to support their concomitant survival and proliferation. Secondly, co-expression of CD49f within the GPI80+ population identifies a subset with reduced short-term myeloid colony-forming activity in semi-solid medium, and greater progeny outputs in both 12-week growth factor-supplemented stromal co-cultures, and in transplanted immunodeficient mice. These findings demonstrate CD49f is a pervasive marker of human HSCs throughout ontogeny and aging. They also reinforce an increasing body of evidence that the molecular maintenance of potent regenerative potential, even in the first HSCs to appear during human development, is adversely affected by exclusive exposure to factors that most strongly stimulate their mitogenesis and even their survival.