The CD34⁺CD38^– phenotype identifies a population in the bone marrow that is enriched in the steady state for hematopoietic stem cells (HSCs). Following ex vivo culture of CD34⁺ cells, HSC content is difficult to measure since committed CD34⁺CD38⁺ progenitors down-regulate CD38 surface expression during culture. In this study, we sought to define the phenotype of human HSCs following ex vivo culture under conditions that support the expansion of human cells capable of repopulating non-obese diabetic/severe combined immunodeficiency (SCID)–repopulating cells (SRCs). Contact coculture of fluorescence-activated cell sorter (FACS)–sorted bone marrow (BM) CD34⁺CD38^– cells with human brain endothelial cells (HUBECs) supported a 4.4-fold increase in CD34⁺CD38^– cells with a concordant 3.6-fold increase in SRCs over 7 days. Noncontact HUBEC cultures and the addition of thrombopoietin, stem cell factor (SCF), and macrophage colony stimulating factor I receptor (Fms)–like tyrosine kinase 3 (Flt-3) ligand supported further increases in CD34⁺CD38^– cells (6.4-fold and 13.1-fold), which correlated with significant increases in SRC activity. Moreover, cell-sorting studies performed on HUBEC-cultured populations demonstrated that SRCs were significantly enriched within the CD34⁺CD38^– subset compared with the CD34^–CD38^– population after culture. These results indicate that human HSCs can be identified and characterized by phenotype following expansion culture. These studies also demonstrate that HUBEC-elaborated soluble factors mediate a unique and potent expansion of human HSCs.