This unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen‐presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique, bone marrow cells are cultured in the presence of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) to yield 5–10 × 10⁶ cells, 60% of which express DC surface markers (e.g., B‐7‐2/CD86). Additional techniques for isolating DCs from mouse spleens or other mouse tissues, as well as from human tissues, are also discussed. Curr. Protoc. Immunol. 86:3.7.1‐3.7.19. © 2009 by John Wiley & Sons, Inc.