All blood cell lineages start from hematopoietic stem cells (HSCs), which were recently shown to represent a heterogeneous group of cells. In mice, Notch signaling promotes the maintenance of “stemness” as well as the expansion of self-renewing HSCs in vitro. Additionally, human CD34⁺ cells were shown to expand in vitro in response to Notch signals. However, it is unclear whether Notch directly affects all HSCs, and whether this role is relevant in vivo. Here, we developed culture conditions that support the maintenance of CD34⁺CD133⁺CD90^lowCD38⁻CD7⁻CD10⁻CD45RA⁻ (CD90^low) cells, phenotypically defined HSCs, as well as 2 early progenitor cells (CD34⁺CD38⁻CD7⁻CD10⁻CD45RA^int [RA^int] and CD34⁺CD38⁻CD7⁻CD10⁻CD45RA^hi [RA^hi]) that were functionally equivalent to multipotent progenitor-2 and lymphoid-primed multipotent progenitor, respectively, found in cord blood. Using a genetic approach, we show that Notch signals were required for HSC preservation, with cultured HSCs being equal to ex vivo HSC cells in their ability to reconstitute immunodeficient mice; however, dnMaml-transduced HSCs were not maintained in vitro. Interestingly, Notch signaling did not appear to be required for the self-renewal of human HSCs in vivo. Our findings support the notion that Notch signals maintain human HSCs in vitro that have hematopoietic-reconstituting ability in vivo and delay the appearance of 2 newly described early progenitor cells.