Retinoic acid-related orphan receptor (ROR) γt+ TCRαβ+ cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3+ RORγt+ T cells, the regulatory counterpart of IL-17+ RORγt+ T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt+ T cells and Th17 cells.