A CD30 ligand (CD30L; CD153) and its receptor, CD30, is a membrane-associated glycoprotein belonging to the TNF superfamily and TNFR superfamily. These were expressed preferentially by activated CD4(+)T cells. In this paper, we show that CD44(low)CD62(hi)CD4(+) T cells from CD30L(-/-) or CD30(-/-) mice exhibited impaired differentiation into Th17 cells but an increased ability to produce IL-2 after in vitro culture under Th17-polarizing conditions. Neutralization with IL-2 by anti-IL-2 mAb partly restored the ability of Th17 differentiation in CD30L(-/-) or CD30(-/-) T cells. Stimulation via CD30L by immobilized anti-CD30L mAb suppressed IL-2 production by CD30(-/-)CD4(+) T cells, indicating that the reverse signal to CD30L is responsible for downregulation of IL-2 production. In vivo Th17 differentiation of CD30L(-/-)CD4(+)CD45RB(high) T cells was also impaired after transfer into SCID mice, whereas CD30L(+/+)CD4(+)CD45RB(high) T cells normally differentiated into Th17 cells in CD30L(-/-) SCID mice. The results of these studies demonstrate that CD30L/CD30 signaling executed by the T-T cell interaction plays a critical role in Th17 cell differentiation, at least partly via downregulation of IL-2 production.