Tumor endothelial marker 8 (TEM8) is uniquely expressed on tumor vasculature but is absent on normal adult vasculature or somatic tissues, making it an attractive target for immunotherapy. The aim of the current study was to induce human cytotoxic T lymphocytes (CTL) with specificity for TEM8 with the end objective of starving tumors by sustained immunological disruption of their vascular supply. Two HLA-A0201 binding peptide epitopes, TEM8207-215 and TEM8298-306 were identified based on predictive algorithms in silico and T2 binding assays in vitro. Individual CTL cultures were established by stimulation of peripheral blood mononuclear cells with peptides in HLA-A2+ healthy donors. CTL induced with each of these peptides mediated lysis of T2 cells pulsed with the respective TEM8 peptide, but not control HIV-RT476-484 peptide, in chromium release assays. However, only CTL induced with TEM8298-306 peptide exhibited specific lysis of an HLA-A2+ TEM8+ HepG2 tumor cells, indicating that TEM8298-306 is a naturally processed peptide epitope. Further characterization of this CTL demonstrated specific secretion of INF-γ and TNF-α per multiplex cytokine arrays. TEM8298-306 peptide does not share sequence homology with peptides derived from other members of the vWF type-A domain family, notably capillary morphogenesis protein-2 which is widely expressed on normal tissues. These results suggest that TEM8298-306 peptide can be safely used in vaccine formulations for tumor immunotherapy.