Flk-1 mutant ES cells do not differentiate into ECs and SMCs in chemically defined medium. Immunostaining of Flk-1 knock-out (Flk-1^-/-), heterozygous (Flk-1^+/-), and wild type (Flk-1+/+) cells at 5.5 days of differentiation with VEGF-A as indicated. (A-C) DAPI staining of cell nuclei. (D-F) PECAM1 staining of EC precursors. (G-I) α-SMA staining for mural precursors. Scale bar, 800 μm. (L) Quantitative RT-PCR of PECAM1 and α-SMA mRNAs and at 8.5 days of differentiation with VEGF-A in serum-free medium of Flk-1 wild-type, heterozygous, and knock-out cells as indicated. (M) Quantitative RT-PCR of PECAM1 and α-SMA mRNAs at 8.5 days of differentiation in FCS of Flk-1 wild-type, heterozygous, and knock-out cells as indicated. The data shown in panels L-M are the results of 3 independent experiments. Error bars indicate standard deviation.