EphB4 deficiency affects the developmental potential of both primitive and definitive hematopoiesis. (A) Primitive erythroid colonies (BFU-Es). Day-5 to -7 EB cells of EphB4^+/– and EphB4^–/– were assayed for their potential to generate primitive erythroid colonies in methylcellulose cultures in the presence of EPO. The right panel is morphology of the representative primitive erythroid colonies from EphB4^+/– ES cells. Data shown represent the average of 4 experiments. Error bars represent standard deviation (P = .003). Original magnification, ×10. (B) Definitive hematopoietic colonies. Day-10 to -12 EB cells of EphB4^+/– and EphB4^–/– were assayed for their potential to generate multilineage hematopoietic colonies in methylcellulose cultures. The right panel is the representative of definitive multilineage hematopoietic colonies from EphB4^+/– ES cells. Error bars represent standard deviation (n = 4, P = .001). (C) Decrease expression of Ter119 and CD45 in EphB4^–/– ES cells. Day-4 and day-10 EB cells of EphB4^+/– or EphB4^–/– were analyzed by flow cytometry. Ter119 is a cell surface marker for erythroid cells and CD45 is a cell surface marker for definitive multilineage hematopoietic cells. Error bars represent standard deviation (n = 2).