Abstract Purpose: The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. Experimental Design: Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell–depleted PBMC. Results: One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327–reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327–specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327–specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01–negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327–specific T cell responses in two of six patients with HLA-A*01–positive melanoma or lung cancer. Conclusion: In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01–restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.