Stat3 inhibition differentially affects primitive erythroblast maturation in vitro. Erythroid progenitor assays were performed on single-cell suspensions of individual, dissociated E8.5 embryos (A) and bone marrow (B,C) cultured in methylcellulose with appropriate media and cytokine supplementation. EryP-CFC (A) colonies were scored after 5 days, d3 BFU-E (B) were scored after 3 days and CFU-E (C) were scored after 2 – 3 days of culture. Primitive erythroblast maturation cultures were performed on cells pooled from dissociated E8.5 embryos (D) while definitive cultures were initiated with ESRE (E). All cultures were treated with DMSO as a vehicle control or 100 μM Stat3 inhibitor, S3I-201. Liquid cultures (D,E) were pretreated with DMSO or S3I-201 for 2 hours prior to EPO stimulation. Definitive erythroblasts are cultured for 2 days, versus 4 days for primitive erythroblasts, because of their more rapid maturation in vitro and in vivo. Images are representative of primitive erythroblasts at days 1 and 4 of culture (D) and definitive erythroblasts at days 0 and 2 of culture (E).