Abstract
Human pluripotent stem cells (hPSCs) provide a powerful platform for generating hematopoietic progenitor cells (HPCs) and investigating hematopoietic development. Here, we present a protocol for maintaining hPSCs and inducing their differentiation into HPCs through the endothelial-to-hematopoietic transition (EHT) on vitronectin-coated plates. We outline steps for evaluating the efficiency of HPC generation and assessing their potential to differentiate into various hematopoietic lineages. This protocol serves as a framework for exploring human hematopoiesis and generating various functional blood cells. For complete details on the use and execution of this protocol, please refer to Shen et al.1 and Qu et al.2.