Bone marrow-derived macrophages (BMM) are primary macrophage cells derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of myeloid progenitors into macrophage/monocyte lineage cells. Mice lacking functional M-CSF have defects in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are cultured in the presence of M-CSF secreted by L929 cells, using L929-conditioned medium. Under these conditions, bone marrow monocyte/macrophage progenitors proliferate and differentiate into a homogeneous population of mature BMMs. The efficiency of differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, BMMs are suitable for various experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages can ingest microbes. A test for the phagocytic efficiency of BMMs is described by exposing them to fluorescently labeled yeast zymosan bioparticles. A method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is also described. Finally, the proliferation of BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division. Materials include L929 cells, CFSE, DAPI, FITC-labeled phalloidin, and various reagents and equipment for cell culture, FACS analysis, and transfection. The protocol outlines the preparation of L929-conditioned medium, isolation and differentiation of BMMs, phagocytosis assays, nucleic acid or protein extraction, and harvesting of BMMs. BMMs are characterized by FACS analysis for Mac-1 and 4/80 surface antigens. Transfection of BMMs using an Amaxa Nucleofector system is described, as well as a cell proliferation assay using CFSE. Troubleshooting tips are provided for common issues such as difficulty in cell dissociation and transfection failure. The discussion highlights the utility of BMMs as a primary cell culture model for studying macrophage functions and gene expression.Bone marrow-derived macrophages (BMM) are primary macrophage cells derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of myeloid progenitors into macrophage/monocyte lineage cells. Mice lacking functional M-CSF have defects in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are cultured in the presence of M-CSF secreted by L929 cells, using L929-conditioned medium. Under these conditions, bone marrow monocyte/macrophage progenitors proliferate and differentiate into a homogeneous population of mature BMMs. The efficiency of differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, BMMs are suitable for various experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages can ingest microbes. A test for the phagocytic efficiency of BMMs is described by exposing them to fluorescently labeled yeast zymosan bioparticles. A method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is also described. Finally, the proliferation of BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division. Materials include L929 cells, CFSE, DAPI, FITC-labeled phalloidin, and various reagents and equipment for cell culture, FACS analysis, and transfection. The protocol outlines the preparation of L929-conditioned medium, isolation and differentiation of BMMs, phagocytosis assays, nucleic acid or protein extraction, and harvesting of BMMs. BMMs are characterized by FACS analysis for Mac-1 and 4/80 surface antigens. Transfection of BMMs using an Amaxa Nucleofector system is described, as well as a cell proliferation assay using CFSE. Troubleshooting tips are provided for common issues such as difficulty in cell dissociation and transfection failure. The discussion highlights the utility of BMMs as a primary cell culture model for studying macrophage functions and gene expression.