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Induced pluripotent stem cells (iPSCs) offer a potentially unlimited source to generate endothelial cells (ECs) for numerous applications. Here, we describe a 7-day protocol to differentiate up to 55 million vascular endothelial cells (viECs) from 3.5 million human iPSCs using small molecules to regulate specific transcription factors. We also describe a parallel-plate flow chamber system to study EC behavior under physiological shear stress. For complete details on the use and execution of this protocol, please refer to Atchison et al. (2020).doi:10.1016/j.xpro.2021.100394 pmid:33796871 pmcid:PMC7995664 fatcat:ozxij657qrcxhkd5ljfsoe32qm