Flow cytometric analysis of Flk-1⁺ cells and Sca-1⁺/c-kit⁺ cells in EBs. (A) The β-gal⁺ cells express Flk-1 in early EB differentiation. ES cells of EphB4^+/– were induced into EB differentiation for 4 days and analyzed by flow cytometry. The β-gal–positive and –negative cells were gated respectively and analyzed for expression of Flk-1 and c-kit. (B-C) Both Flk-1⁺ and Sca-l⁺/c-kit⁺ cells decreased in EphB4^–/– EBs. Day-4 and day-9 EB cells from either EphB4^+/– or EphB4^–/– ES cells were analyzed for Flk-1 (B) and Sca-1/c-kit (C) expression by flow cytometry. Dead cells were gated out by a DNA dye, DAPI, staining. Flow cytometric data are representative from 3 independent experiments.