Lack of ALAS-E affects erythroid differentiation of ES cells at late stage. EBs formed from the wild-type (a) and ALAS-E(-) mutant ES cells (b) were dissociated with collagenase and stained with benzidine. Note the presence of dark blue cells in the wild-type EBs, which are positive to the benzidine staining. The wild-type (c) and mutant ES cells (d) were also cocultured with OP9 stroma cells and colonies formed were stained with benzidine. Hematopoietic cells obtained from the two-step coculture culture system with OP9 cells were then analyzed. The expression of TER119 was analyzed by FACS. Comparable numbers of TER119-positive cells were floated from ALAS-E(-) mutant ES cell culture (f) as does from the wild ES cell culture (e). However, no benzidine staining-positive cells were observed in the hematopoietic cells from the mutant ES cells (h), while a number of benzidine-positive cells were observed in the floating hematopoietic cells from the wild-type ES cells (g).