Brain microvascular endothelial cells (BMECs) constitute the blood–brain barrier (BBB), which prevents the transfer of substances into the brain. Recently, in vitro BBB models using human-induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been created. However, it is suggested that iBMELCs differentiated by the existing methods are different from the BMECs that occur in vivo. This study aimed to establish iBMELCs generated via human iPS cell-derived endothelial progenitor cells (iEPCs) (E-iBMELCs). Expanded and cryopreserved iEPCs were thawed and differentiated into mature endothelial cells under various conditions. Intercellular barriers were significantly enhanced in E-iBMELCs using a B-27 supplement, transforming growth factor-β receptor inhibitor, and laminin 511 fragment. Expression of the endothelial cell markers was higher in the E-iBMELCs generated in this study compared with conventional methods. In addition, E-iBMELCs expressed P-glycoprotein. E-iBMELCs developed in this study will significantly contribute to drug discovery for neurodegenerative diseases and might elucidate the pathogenesis of neurodegenerative diseases associated with BBB disruption.