This protocol outlines the isolation and characterization of bone marrow-derived macrophages (BMMs) and their applications in various experimental studies. BMMs are primary macrophage cells derived from bone marrow cells in the presence of growth factors, particularly macrophage colony-stimulating factor (M-CSF). The protocol details the preparation of L929-conditioned medium, which is used to culture and differentiate bone marrow cells into BMMs. The efficiency of differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of surface antigen expression. Once differentiated, BMMs can be used for morphological, gene expression, and physiological studies, including phagocytic activity, gene delivery, and cell proliferation assays. The protocol also includes troubleshooting tips for common issues such as trypsinization, transfection, and FACS analysis. BMMs are highlighted as a valuable model for studying immune functions and gene function in vitro due to their homogeneity, proliferative capacity, and long lifespan.This protocol outlines the isolation and characterization of bone marrow-derived macrophages (BMMs) and their applications in various experimental studies. BMMs are primary macrophage cells derived from bone marrow cells in the presence of growth factors, particularly macrophage colony-stimulating factor (M-CSF). The protocol details the preparation of L929-conditioned medium, which is used to culture and differentiate bone marrow cells into BMMs. The efficiency of differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of surface antigen expression. Once differentiated, BMMs can be used for morphological, gene expression, and physiological studies, including phagocytic activity, gene delivery, and cell proliferation assays. The protocol also includes troubleshooting tips for common issues such as trypsinization, transfection, and FACS analysis. BMMs are highlighted as a valuable model for studying immune functions and gene function in vitro due to their homogeneity, proliferative capacity, and long lifespan.