As T cell activation leads to downregulation of T cell receptor (TCR) and coreceptor CD8, we developed a novel FACS-based sorting method to enrich activated antigen-specific CD8+ T cells. Using multiple established or low percentage T cell cultures, with either single antigen specificity or multiple influenza A virus antigen specificities, we have optimized the sorting method for T cell activation time and stimulating antigen dose. We have also sorted various numbers of antigen-specific CD8+ T cells into 96-well plates to demonstrate these T cells are capable of expanding into nearly pure CD8+ T cell lines. Our approach has the advantage of sorting antigen-specific T cells without knowing their specific antigenic epitopes or restricting HLA. We believe this method can be very helpful for successfully establishing CD8+ T cell lines for various purpose, including immunotherapy.