Objective:As Th17 cells lack of specific surface marker,it is difficult to achieve highly purified population of Th17 cells. We sought to obtain highly purified population of Th17 cells by adjusting the concentration of cytokines and different cultivation methods. Methods:CD4+ CD44- naive T cells were separated from C57BL/6J mice by magnetic-activated cell sorting (MACS). Different inducing systems were used for testing the effect of different cytokines and culture methods. The ratio of Th17 cells was analyzed by flow cytometry,the expressions of IL-17,ROR-γt,IFN-γ,IL-4 and FoxP3 mRNA were measured by qRT-PCR method and the IL-17 concentration in cell culture supernatant was detected by ELISA. Results:Th17 cells were induced by TGF-β,IL-6 and IL-23,the ratio of Th17 increased by adding TNF-α,IL-1β,anti-IFN-γ and anti-IL-2. The use of U-type 96 wells plate was significantly better than the 24 wells plate in culturing Th17 cells. Th17 cells showed steady growth when cultured in U-type 96 wells plate and the ratio of Th17 cells was significantly increased [(91.85 ± 1.05)%]. The expressions of IL-17 and ROR-γt mRNA were up-regulated compared with that of Naive T cells (P < 0.001),however,IFN-γ,IL-4 and Foxp3 mRNA were decreased (P < 0.001). The concentration of IL-17 in cell culture supernatant was significantly increased (P < 0.001). Conclusion:The regulated method can generate a highly purified population (>90%) of Th17 cells.