Background: Adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) is an effective approach for the treatment of metastatic melanoma, with objective response rates of approximately 50%. An increased understanding of the factors contributing to resistance to this promising therapy is needed. Methods: To determine the role of epigenetic mechanisms in TIL responses, cell infusion products from patients undergoing TIL ACT were separated by magnetic labeled antibodies into CD4 and CD8 populations, and assessed by an Illumina 450K DNA methylation array and chromatin immunoprecipitation of acetylated histone 3 with sequencing. Patient tumor samples were also assessed on a Nanostring platform for epigenetic gene and miR expression. The percentage of CD4 and CD8 T cells in TIL were assessed by flow cytometry. Results: Significant differences in DNA methylation of genes controlling CD4 lineage (CD4, CD8, RUNX3, XCL2, ZBTB7B) were found between responders (R) and non-responders (NR) (p < 0.05). Using a CD4/CD8 differentiation signature, principal component analysis (PCA) revealed clustering based on response, with NR CD8 T cells more closely resembling CD4 T cells (p < 0.05). PCA of acetylated histone 3 also showed differences between R vsNR (p < 0.05). Nanostring analysis of tumor fragments showed decreased expression in NR in several epigenetic regulatory genes including DNA methyltransferases (DNMT1, DNMT3A) and histone deacetylases (HDAC8, SIRT4)(p < 0.05). MiR-148 and miR-185, known regulators of DMNT1, were increased in accordance with the patterns seen for DNMT1, a known regulator of CD4/CD8 lineage commitment. Higher levels of CD4/CD8 double positive TILs were found in NR infusion products. Conclusions: These results demonstrate that resistance to TIL therapy is characterized by altered DNA methylation and histone acetylation patterns accompanied by differences in genes regulating these marks. It is hypothesized that epigenetic dysregulation of CD4/CD8 lineage impairs the therapeutic efficacy of TIL therapy. These data highlight an unexplored mechanism of T-cell dysfunction characterizing resistance to TIL ACT.