Th17 cells are pro-inflammatory cells that are characterized by the production of their signature cytokine, IL-17. Although they are thought to have arisen to protect against extracellular bacteria and fungi they have been shown to mediate autoimmune diseases such as EAE and psoarisis. Notch protein is a cell-surface receptor that has been widely conserved among species. It plays an essential role in determining multiple cell fates. More recently, it has been implicated in regulating peripheral CD4+ T-cell responses. In these studies, we report that blockade of Notch signaling significantly down-regulates the production of IL-17 and associated cytokines in both mouse and human in-vitro polarized Th17 cells, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies as well as chromatin immunoprecipitation, that IL-17 and RORt are direct transcriptional targets of Notch signaling in Th17 cells, with Notch 1 being the responsible Notch family member important in regulating the differentiation of human Th17 cells. In-vivo inhibition of Notch signaling reduced IL-17 production and Th17 mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. In addition, by using Notch1 and Notch3 knockout mice, we have shown that Notch 3 is the Notch family member that is essential for murine Th17 differentiation. We have also investigated noncanonical Notch signaling in Th17 cells by using CD4+ T-cells from CSL/RBP-Jk knockout mice. Based on data obtained, we have concluded that canonical Notch signaling is dispensable in Th17 responses. Thus, this study highlights the importance of different Notch family members in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.