The use of T cell receptor (TCR) gene-modified T cells in adoptive cell transfer has had promising clinical success, but often, simple preclinical evaluation does not necessarily accurately predict treatment efficacy or safety. Preclinical studies generally evaluate one or a limited number of type 1 cytokines to assess antigen recognition. However, recent studies have implicated other "typed" T cells in effective anti-tumor/viral immunity, and limited functional evaluations may underestimate cross-reactivity. In this study, we use an altered peptide ligand (APL) model and multi-dimensional flow cytometry to evaluate polyfunctionality of TCR gene-modified T cells. Evaluating six cytokines and the lytic marker CD107a on a per cell basis revealed remarkably diverse polyfunctional phenotypes within a single T cell culture and among peripheral blood lymphocyte (PBL) donors. This polyfunctional assessment identified unexpected phenotypes, including cells producing both type 1 and type 2 cytokines, and highlighted interferon γ (IFNγ) antigen-reactive populations overlooked in our previous studies. Additionally, APLs skewed functional phenotypes to be less polyfunctional, which was not necessarily related to changes in TCR-peptide-major histocompatibility complex (pMHC) affinity. A better understanding of gene-modified T cell functional diversity may help identify optimal therapeutic phenotypes, predict clinical responses, anticipate off-target recognition, and improve the design and delivery of TCR gene-modified T cells.