Antigen-specific T cells represent a potential therapeutic avenue for a variety of conditions, but current approaches for generating such cells for therapeutic purposes are limited. In this study, we established iPSCs from mature cytotoxic T cells specific for the melanoma epitope MART-1. When cocultured with OP9/DLL1 cells, these iPSCs efficiently generated TCRβ⁺CD4⁺CD8⁺ double positive (DP) cells expressing a T cell receptor (TCR) specific for the MART-1 epitope. Stimulation of these DP cells with anti-CD3 antibody generated a large number of CD8⁺ T cells, and more than 90% of the resulting cells were specific for the original MART-1 epitope. Stimulation of the CD8⁺ T cells with MART-1 antigen-presenting cells led to the secretion of IFNγ, demonstrating their specific reactivity. The present study therefore illustrates an approach for cloning and expanding functional antigen-specific CD8⁺ T cells that might be applicable in cell-based therapy of cancer.