Th17 cells (interleukin-17 [IL-17]-secreting T helper cells) have been implicated in the pathogenesis of rheumatoid arthritis and other autoimmune diseases, but the soluble factors that influence human Th17 differentiation have yet to be fully elucidated. This study was undertaken to investigate the hypothesis that the cytokines secreted by human peripheral blood mononuclear cells (PBMCs) in response to a subset of Toll-like receptor (TLR) ligands would influence Th17 polarization.Supernatants from human PBMCs treated with a panel of TLR agonists were tested for their ability to induce de novo IL-17 production in naive T helper cells. Multiplex cytokine analysis was used to identify candidate cytokines for subsequent blocking and sufficiency experiments.Conditioned media from PBMCs stimulated with TLR-4 or TLR-8/7 agonists, but not from those stimulated with TLR-2/1, -3, or -9 agonists, evoked robust secretion of IL-17 by T helper cells, independent of coculture with antigen-presenting cells. Multiplex analysis of 22 cytokines and chemokines identified a 6-factor cytokine signature that significantly correlated with IL-17-inducing activity. T cell activation in the presence of recombinant IL-1beta, IL-6, and IL-23 reconstituted robust IL-17 production, and this was enhanced by transforming growth factor beta (TGFbeta). IL-6 suppressed the expression of forkhead box P3 and reversed TGFbeta-mediated inhibition of T cell proliferation, but did not trigger IL-17 secretion. IL-17 production was completely abrogated by anti-IL-1 or IL-1 receptor antagonist and partially inhibited by anti-IL-6, anti-IL-2, or exogenous retinoic acid, but not by anti-tumor necrosis factor alpha. IL-1beta and IL-6 independently induced IL-21 secretion, but the presence of IL-21 alone was not sufficient for IL-17 production.These results indicate that ligation of a subset of TLRs generates proinflammatory cytokines that combine to potentiate human Th17 differentiation.