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This protocol describes the use of SB431542, a TGF-beta inhibitor molecule, for differentiation and robust expansion of hESCs into endothelial cells. In addition, it contains a purification step by FACS or MACS. The authors used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to generate transgenic hESC cells. These transgenic cells were further used to screen for factors that promote vascular commitment.