ABSTRACTWe describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34 + (10 5 cells/mL) or light-density (10 6 cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10 \ - 6 M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated |ms1\N2 \x 10 7 erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90\%) of CD45 l o w /glycophorin (GPA) n e g /CD71 1 o w cells at day 7, 50\N60\% of which became CD45 n e g /GPA + /CD71 h i g h by days 15\N20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (\g90\% benzidine p o s and CD45 n e g /GPA + /CD71 m e d i u m ) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.