The rapid development of genomics and proteomics has accelerated the discovery of antigens that play a role in host-tumor interaction and can be potentially targeted in tumor immunotherapy. Several independent approaches to characterize such antigens and identify the relevant epitopes have been developed. However, the detection, expansion, and characterization of antigen-specific T cells are essential steps common to all strategies. Efficient identification of epitopes, in particular in a preclinical setting, is often hampered by lack of significant numbers of antigen presenting cells at sufficient purity that readily expand low-frequency T-cell precursors. Using the cylins as a model family of self-tumor antigens, we show that CD40-activated primary human B cells can be used very efficiently to identify novel epitopes and characterize such tumor antigen-specific T cells.