The monocyte capacity to differentiate into dendritic cells (DCs) was originally demonstrated by human in vitro DC differentiation assays that have subsequently become the essential methodologic approach for the production of DCs to be used in DC-mediated cancer immunotherapy protocols. In addition, in vitro DC generation from monocytes is a powerful tool to study DC differentiation and maturation. However, whether DC differentiation from monocytes occurs in vivo remains controversial, and the physiologic counterparts of in vitro monocyte-derived DCs are unknown. In addition, information on murine monocytes and monocyte-derived DCs is scarce. Here we show that mouse bone marrow monocytes can be differentiated in vitro into DCs using similar conditions as those defined in humans, including in vitro cultures with granulocyte-macrophage colony-stimulating factor and interleukin 4 and reverse transendothelial migration assays. Importantly, we demonstrate that after in vivo transfer monocytes generate CD8- and CD8+ DCs in the spleen, but differentiate into macrophages on migration to the thoracic cavity. In conclusion, we support the hypothesis that monocytes generate DCs not only on entry into the lymph and migration to the lymph nodes as proposed, but also on extravasation from blood and homing to the spleen, suggesting that monocytes represent immediate precursors of lymphoid organ DCs.