FLT3 activation enhances hematopoietic differentiation from hESCs. (A) Schematic of the hematopoietic differentiation of hESCs and end point analyses (top) and representative flow cytometry dot plots displaying how hemogenic precursors (CD45⁻ CD31⁺), primitive blood cells (CD45⁺ CD34⁺), and total blood cells (CD45⁺) are identified (bottom). (B) PCR confirming the presence of either FLT3-TKD or FLT3-ITD mutations in transgenic day 15 hEBs. (C) Specification into hemogenic precursors is not significantly affected by FLT3 activation throughout EB development. However, FLT3 activation enhances hESC differentiation into primitive blood cells (D) and total blood cells (E). (F) CFU readout from d15 hEBs confirms a significant increase in hematopoietic potential in hESCs expressing either FLT3 mutation. Data are presented as mean ±SEM for 6 independent experiments. FLT3 mutation–expressing hESC-derived hematopoietic cells do not show stable in vitro replating efficiency in secondary CFU assays. Right panels depict representative CFU colonies.