Hematopoietic stem cells (HSCs) have been extensively characterized based on functional definitions determined by experimental transplantation into lethally irradiated mice. In mice, HSCs are heterogeneous with regard to self-renewal potential, in vitro colony-forming activity, and in vivo behavior. We attempted prospective isolation of HSC subsets with distinct properties among CD34^−/low c-Kit⁺Sca-1⁺Lin⁻ (CD34⁻KSL) cells. CD34⁻KSL cells were divided, based on CD150 expression, into three fractions: CD150^high, CD150^med, and CD150^neg cells. Compared with the other two fractions, CD150^high cells were significantly enriched in HSCs, with great self-renewal potential. In vitro colony assays revealed that decreased expression of CD150 was associated with reduced erythroblast/megakaryocyte differentiation potential. All three fractions were regenerated only from CD150^high cells in recipient mice. Using single-cell transplantation studies, we found that a fraction of CD150^high cells displayed latent and barely detectable myeloid engraftment in primary-recipient mice but progressive and multilineage reconstitution in secondary-recipient mice. These findings highlight the complexity and hierarchy of reconstitution capability, even among HSCs in the most primitive compartment.