Th17 cells represent a T helper cell subset with major implications for the pathogenesis of autoimmune diseases and the clearance of extracellular bacteria and fungi. The in vitro generation of human Th17 cells has been subject to many debates concerning the minimal cytokine requirements for IL-17 induction. This is partly due to the low Th17 cell priming efficiencies that have been reported so far for human as compared to murine T cells. In addition, human T helper cell priming is primarily performed using polyclonal stimulation even though it has recently been reported that cytokine requirements for the generation of Th17 cells may differ depending on the microbial antigen specificities of naïve T cells. Here, we present a detailed procedure on how to efficiently generate microbe-specific Th17 cells from naïve T helper cells.