Fig. 5. Expression of FLK1 and GFP in differentiating Mixl1 wild-type (w/w), heterozygote (GFP/w) and null (GFP/GFP) ES cells. (A) Time course of FLK1 and GFP expression showing that the onset of FLK1 expression is delayed and that the frequency of FLK1-positive cells was reduced in Mixl1-deficient ES cells. (B,C) Summary of data (n=3-6) demonstrating a lower percentage of FLK1⁺ cells (B) and a reduction in the ratio of double-positive GFP⁺FLK1⁺ to GFP⁺ cells (C) in day 4 EBs derived from two independent Mixl1-deficient ES cell lines compared with their heterozygote counterparts. The Mixl1-null lines M916 and M3C5 were derived from Mixl1-heterozygous ES cell lines M147 and M114, respectively. Mixl1^w/w is the parental ES cell line. The error bars represent 1 s.d. and the P values indicated were derived using a two-tailed t-test.