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Summary: Human induced pluripotent stem cell (hiPSC)-derived macrophages provide a valuable tool for disease modeling and drug discovery. Here, we present a protocol to generate functional macrophages from hiPSCs using a feeder-free hematopoietic differentiation technique. We describe steps for preparing hiPSCs, mesodermal differentiation, hematopoietic commitment, and macrophage differentiation and expansion. We then detail assays to characterize their phenotype, polarization, and phagocytic functions. The functional macrophages generated here could be used to generate organoids for disease modeling and drug discovery studies.For complete details on the use and execution of this protocol, please refer to Jeong et al.1 and Heo et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.