Fig. 5. VEGF is not necessary for FLK1⁺ cell production, but is necessary for the generation of BL-CFCs. (A) Arnt^+/+, Arnt^–/–, Vegf^+/– and Vegf^–/– ES clones were differentiated for 3 days under normoxic (21% O₂) or hypoxic (3% O₂) conditions and replated for their hemangioblast potential. Error bars represent SEMs of triplicate cultures. (B) Generation of BL-CFCs in Arnt^+/+ cultures is inducible with the addition of VEGF (5 and 10 ng/ml) during early time points (day 1.5-2.5), but exogenous VEGF does not rescue the Arnt^–/– cultures. (C) VEGF and bFGF do not rescue FLK1⁺ cell production in Arnt^–/– EB cultures; Vegf^+/– and Vegf^–/– cells exhibit no FLK1⁺ deficiency and FLK1⁺ cell numbers can be further induced with the addition of VEGF and bFGF, or by hypoxia treatment.