Impaired BL-CFC formation of EphB4-deficient ES cells. (A) Examples of blast colony (BL-CFC) and secondary EB from EphB4^+/– ES cells. BL-CFCs can be readily recognized as loose clusters compared with the compact secondary embryoid bodies. Original magnification, × 40. (B) Day-3 EBs of EphB4^+/– and EphB4^–/– ES cells were collected, and the cells were dissociated and assayed for their potential to generate BL-CFCs and secondary EB in methylcellulose cultures. Error bars represent standard deviation. P values were calculated with student t test. Data shown represent the average of 4 experiments. (C) Kinetic analysis of BL-CFC formation. The cells from EBs at indicated times were analyzed for BL-CFC generation. Data shown represent the average of 4 experiments. Error bars represent standard deviation. P values were calculated with Student t test. (D) RT-PCR analysis of developmental potential of the BL-CFCs. Day-3 EBs of EphB4^+/– and EphB4^–/– ES cells were used to generate BL-CFCs. Blast colonies from either EphB4^+/– or EphB4^–/– were transferred into matrigel-coated plates and cultured in the presence of growth factors known to support both hematopoietic and endothelial cells. Following 3 to 4 days in culture, nonadherent hematopoietic and adherent endothelial cells were harvested for RT-PCR analysis of lineage specific genes.