Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.