This study was to explore a potential role of epithelium-derived cytokines in Th17 differentiation. Th17 induction was evaluated by murine CD4(+) T cells treated with different combinations of five inducing cytokines, or conditioned media of human corneal epithelial cells (HCECs) exposed to a variety of stimuli. Th17 differentiation was determined by measuring Th17 associated molecules, IL-17A, IL-17F, IL-22, CCL-20, and STAT3 at mRNA and protein levels, and numbers of IL-17-producing T cells by real-time PCR, and cytokine immunobead and ELISPOT assays, respectively. IL-23 was the strongest inducer for expanding Th17 cells in the presence of TGF-beta1 + IL-6; and IL-1beta was the strongest Th17 amplifier in the presence of TGF-beta1 + IL-6 + IL-23. These inducing cytokines were found to be significantly stimulated in HCECs challenged by hyperosmotic media (450 mOsM), microbial components (polyI:C, flagellin, R837, and other TLR ligands) and TNF-alpha. Interestingly, when incubated with conditioned media of HCECs irritated by polyI:C or TNF-alpha, CD4(+) T cells displayed increased mRNA levels of IL-17A, IL-17F, IL-22, CCL-20, and STAT3, increased IL-17 protein in the supernatant, and increased numbers of IL-17-producing T cells (Th17 cells). These findings demonstrate for the first time that Th17 differentiation can be promoted by cytokines produced by corneal epithelium that are exposed to hyperosmotic, microbial, and inflammatory stimuli.