Endothelial development in EphB4-deficient EBs is impaired. (A) EphB4^–/– EBs display a less well defined vasculogenic morphology. ES cells of EphB4^+/– (left) and EphB4^–/– (right) were induced for vasculogenesis in the presence of VEGF, bFGF, EPO, and IL-6 in methylcellulose for 10 to 11 days. EBs were then transferred in collagen cultures for 3 to 6 days to develop sprouting EBs. (B) Capillary-like structures of EphB4^+/– EBs were CD31⁺ by immunohistochemistry. EB cultures in collagen matrix were dehydrated and fixed in a methanol-acetone solution and stained with PE-conjugated antimouse PECAM1 (CD31; Pharmingen). Original magnification, × 4. (C) Total numbers of vascular-like EBs were significantly decreased in EphB4^–/^– EBs. EB in collagen matrix were analyzed for sprouting EB induction. Total EBs and capillary-like EBs (vascular EBs) were counted in EphB4^+/– and EphB4^–/– cultures. Error bars represent standard deviation (n = 9).