Abstract TH17 cells play an essential role in regulating host defense and exacerbating various autoimmune diseases, but their role in tumor immunity remains incompletely elucidated. We found that CD28 abrogates TH17 expansion and function, while the inducible costimulator (ICOS) augments the generation of polyfunctional TH17 cells. Of clinical importance, ICOS-expanded TH17 cells were superior at killing human tumors compared to cells expanded with CD28. Our objective is to identify how ICOS regulates the differentiation and optimal expansion of TH17 cells that mediate tumor regression. We have polarized human CD4+ T cells towards TH17 with beads expressing anti-CD3 and CD28 and/or ICOS agonists. ICOS costimulation promotes the long-term generation of TH17 cells, determined by CD161 and CCR6 expression and IL-17A production. By sorting peripheral blood cells based on these two receptors, we identified a subset of cells that nearly all produce IL-17 when stimulated with an ICOS agonist. This innovative approach allows us to study the basic biology of IL-17-producing cells and affords us the ease of exclusively enriching them for adoptive immunotherapy for cancer. We are now uncovering new transcription factors uniquely regulated by ICOS that distinctly impact TH17 cells’ phenotype and functional fate. Understanding how ICOS signaling and optimal culture conditions promote TH17 cell function will be instrumental in advancing T cell-based therapies for cancer and infectious diseases.