Dendritic cells (DCs) are potent antigen‐presenting cells (APC) that are important in the initiation and control of cellular immune responses. Commonly used in T cell–stimulation experiments, DCs are typically “matured” in vitro with microbial products or proinflammatory cytokines, and then loaded with antigens from any number of sources, including peptides, whole proteins, cell lysates, RNA, microbes, or killed tumor cells. This unit presents a simple and commonly used method for the generation of mature human dendritic cells—differentiating them from peripheral blood monocytes.