Day 7 to 20 hEB cells contain clonogenic progenitors for both primitive and definitive erythropoiesis. Erythroid colonies with a “brilliant red” hemoglobinization from day 9 hEB-derived BFU-e-P (Ai; magnification × 100) and EryP (ii; magnification × 100) containing nucleated primitive erythrocytes were generated from day 7 to 15 day hEBs. Erythroid colonies containing a brownish, “salmon-red” hemoglobinization were generated from day 12 to 20 day hEBs ([B] BFU-e-D, CFU-e-D). Colonies (3 to 5 pooled) were picked from semisolid medium for staining or FACS analyses. Wright stains from multiclustered BFU-e-P (Aiii; magnification × 600) revealed an increased abundance of erythroblasts, while more mature EryP (iv; magnification × 600) contained primarily differentiated nucleated erythrocytes. Primitive erythroblast (Bl) colonies predominate from day 9 to 12 hEBs differentiate directly into nucleated erythrocytes positive for hemoglobin by benzidine staining (C, top; magnification × 400). Erythroblast colonies stain brightly positive for fetal hemoglobins by erythrosin-B K-B stains (C, bottom; magnification × 200). (Inset) Positive control K-B staining from maternal peripheral blood with 10% fetal erythrocyte cells. (D) Kinetic analysis of primitive and definitive erythroid CFCs. Shown is a representative analysis of 3 independent experiments. Although all types of erythroid CFCs had surface expression of CD71⁺/GlyA⁺ cells (F) and expressed various levels fetal hemoglobin (HbF), only day 12 to 20 BFU-e-D and CFU-e-D expressed adult hemoglobin A (HbA). Day 15 to 20 BFU-e-D or CFU-e-D often contained brownish, lysed cells. Coexpression of both HbF and HbA in BFU-e-D from CD34⁺ cord blood (CB) progenitors was used as a positive control ([F] CB BFU-e).