BACKGROUND: Requirements for human B lymphopoiesis are still poorly understood, and that has hampered investigation of differentiation events. For example, there are few cell surface antigens that can be used as milestones of lineage progression. The CD10 ectoenzyme is one such marker and has been used to define CLP, but we found substantial tissue specific variations in CD10 levels, and there was no information about how that corresponded to differentiation options. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to use recently developed culture methods to assess the nature and differentiation potential of progenitors sorted according to CD10 density from umbilical cord blood (CB), adult bone marrow (BM) or G-CSF mobilized peripheral blood (PB). Many CD34(+) cells in BM express high levels of CD10, while low or low/negative CD10 densities were found on CD34(+) cells in CB or G-CSF mobilized PB, respectively. The relative abundance of CD10(Lo) versus CD10(Hi) cells only accounts for some CB versus BM differences. Almost all of the CD34(+) CD10(Hi) cells expressed CD19 and lymphocyte transcription factors and corresponded to loss of myeloid potential. A high degree of immunoglobulin D(H)-J(H) gene rearrangements was characteristic only of the CD10(Hi) subset. In contrast, the CD34(+) CD10(Lo) progenitors efficiently produced plasmacytoid and conventional dendritic cells as well as myeloid cells. These findings suggest a positive correlation between CD10 density and degree of differentiation. Although freshly isolated CD34(+) CD10(Hi) cells were in cycle, those from CB or BM expanded poorly in culture, suggesting regulators of populations remain to be discovered. CONCLUSIONS/SIGNIFICANCE: Steps in human B lymphopoiesis have not been sufficiently studied, and we now show that increased CD10 expression corresponds to differentiation potential and stage. CD34(+) CD10(Hi) progenitors are obviously in the B lineage but may have progressed beyond the point where they can be expanded in culture.