Bone marrow-derived macrophages (BMDMs) mimic several properties of macrophages, such as phagocytosis and antigen processing and presentation, and may be used to study and characterize macrophage biology in response to specific stimuli. BMDMs are generated through isolation of cells from murine femur and tibia and cultured with L929-conditioned media, which contain factors essential for differentiation into macrophages. This protocol details the steps of retrieving bone marrow from mice, collecting L929-condition media, and culturing bone marrow cells to differentiate into macrophages.