The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is essential for the initiation of CD8⁺ T cell responses. In vivo, cross-presentation is mainly carried out by specific dendritic cell (DC) subsets through an adaptation of their endocytic and phagocytic pathways. Here, we summarize recent advances in our understanding of the intracellular mechanisms of cross-presentation and discuss its role in immunity and tolerance in the context of specialization between DC subsets. Finally, we review current strategies to use cross-presentation for immunotherapy.