Macrophages are found in tissues throughout the body and are important immune cells, however, these tissue macrophages are difficult to collect and study. Therefore, the ability to differentiate macrophages from peripheral blood precursors is an important research tool. Macrophage differentiation has been well studied in humans, but differentiation in the non-human primate is poorly characterized. Using human models is not always feasible for invasive experimental studies and, therefore, developing reliable protocols for the non-human primate model is important. We describe a method to differentiate macrophages in vitro in the rhesus monkey by culturing adherent peripheral blood mononuclear cells for five days in RPMI-1640 supplemented with 1% human serum, M-CSF, and IL-1beta. The resulting cells had a distinct macrophage phenotype, the ability to secrete cytokines in response to LPS, and antigen uptake and processing capabilities.