Antigen cross-presentation is an adaptation of the cellular process of loading MHC-I molecules with endogenous peptides during their biosynthesis within the endoplasmic reticulum. Cross-presented peptides derive from internalized proteins, microbial pathogens, and transformed or dying cells. The physical separation of internalized cargo from the endoplasmic reticulum, where the machinery for assembling peptide-MHC-I complexes resides, poses a challenge. To solve this problem, deliberate rewiring of organelle communication within cells is necessary to prepare for cross-presentation, and different endocytic receptors and vesicular traffic patterns customize the emergent cross-presentation compartment to the nature of the peptide source. Three distinct pathways of vesicular traffic converge to form the ideal cross-presentation compartment, each regulated differently to supply a unique component that enables cross-presentation of a diverse repertoire of peptides. Delivery of centerpiece MHC-I molecules is the critical step regulated by microbe-sensitive Toll-like receptors. Defining the subcellular sources of MHC-I and sites of peptide loading during cross-presentation remain key challenges. Expected final online publication date for the Annual Review of Immunology Volume 36 is April 26, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.