Macrophages are specialized phagocytes that display a variety of important functions for the host immune system. They are particularly important for the recognition of exogenous and endogenous danger signals, forming the defensive front line as part of innate immune response. As such, murine macrophages are commonly used for in vitro cell-based assays examining the mechanisms of innate immune activation, which can require the ongoing breeding and housing of a large number of genetically modified mouse strains. Here, we describe a robust protocol for the generation of immortalized bone marrow-derived macrophages (iBMDMs) from primary murine bone marrow cells. We further provide general protocols for harvesting, freezing, and thawing of bone marrow cells, maintaining iBMDMs in culture and generation of monoclonal iBMDM populations by single-cell cloning.