Diamond-Blackfan anemia (DBA) is a disorder characterized by a selective defect in erythropoiesis. Delineation of the precise defect is hampered by a lack of markers that define cells giving rise to erythroid burst- and erythroid colony-forming unit (BFU-E and CFU-E) colonies, the clonogenic assays that quantify early and late erythroid progenitor (EEP and LEP) potential, respectively. By combining flow cytometry, cell-sorting, and single-cell clonogenic assays, we identified Lin⁻CD34⁺CD38⁺CD45RA⁻CD123⁻CD71⁺CD41a⁻CD105⁻CD36⁻ bone marrow cells as EEP giving rise to BFU-E, and Lin⁻CD34^+/−CD38⁺CD45RA⁻CD123⁻CD71⁺CD41a⁻CD105⁺CD36⁺ cells as LEP giving rise to CFU-E, in a hierarchical fashion. We then applied these definitions to DBA and identified that, compared with controls, frequency, and clonogenicity of DBA, EEP and LEP are significantly decreased in transfusion-dependent but restored in corticosteroid-responsive patients. Thus, both quantitative and qualitative defects in erythroid progenitor (EP) contribute to defective erythropoiesis in DBA. Prospective isolation of defined EPs will facilitate more incisive study of normal and aberrant erythropoiesis.