FLT3-activating mutations block hematopoietic differentiation from MLL-AF4-hESCs. (A) RT-PCR confirming stable expression of MLL-AF4 in transgenic day 15 hEBs. (B) PCR confirming the presence of either FLT3-TKD or FLT3-ITD mutations in transgenic day 15 hEBs. FLT3-activating mutations only impair late specification into hemogenic precursors of MLL-AF4–expressing hESCs (C) but completely block differentiation of MLL-AF4–expressing hESCs into primitive blood cells (D) and total blood cells (E). (F) CFU readout from d15 hEBs confirms a significant decrease in hematopoietic potential in hESCs coexpressing MLL-AF4 and FLT3 mutations. Data are presented as mean ± SEM for 9 independent experiments. MLL-AF4/FLT3 mutation–expressing hESC-derived hematopoietic cells do not show stable in vitro replating efficiency in secondary CFU assays.