Cross-presentation is increasingly recognized as a key mechanism by which specific antigen-presenting cells (APCs) activate the cellular immune system against virally infected or neoplastic cells. These APCs have the capacity to acquire exogenous proteins by phagocytosis or endocytosis, derive antigenic peptides, and then cross-present them in the context of class I major histocompatibility complex molecules. Because APCs provide both an antigenic stimulus and costimulatory signals, they can effectively activate naive CD8+ T lymphocytes, resulting in a brisk cellular immune response. The precise cellular pathways that permit an exogenous antigen to be presented in the context of class I major histocompatibility complex is the focus of intense investigation that has illuminated our understanding of the cell biology of APCs. This article reviews our understanding of how APCs cross-present antigen, illustrating how this process differs from the canonical pathways of antigen presentation. We define the central players required for cross-presentation and then focus on recent studies that reveal the molecular mechanisms underlying this phenomenon. Understanding these mechanisms will likely inform the development of effective cell-based vaccines and other cellular immunotherapies and is therefore of interest to practitioners of transfusion medicine.