Notch signaling pathway plays an important role in T cell differentiation. Delta-like ligand (Dll)4, one of five known Notch ligands, has been implicated in regulating Th2 cell differentiation in animal models of human diseases. However, the role of Dll4 in Th1/Th17-mediated autoimmune diseases remains largely unknown. Using an anti-Dll4 blocking mAb, we show that neutralizing Dll4 during the induction phase of experimental autoimmune encephalomyelitis in C57BL/6 mice significantly increased the pool of CD4(+)Foxp3(+) regulatory T cells (Treg) in the periphery and in the CNS, and decreased the severity of clinical disease and CNS inflammation. Dll4 blockade promoted induction of myelin-specific Th2/Treg immune responses and impaired Th1/Th17 responses compared with IgG-treated mice. In vitro, we show that signaling with recombinant Dll4 inhibits the TGF-β-induced Treg development, and inhibits Janus kinase 3-induced STAT5 phosphorylation, a transcription factor known to play a key role in Foxp3 expression and maintenance. Depletion of natural Treg using anti-CD25 Ab reversed the protective effects of anti-Dll4 Ab. These findings outline a novel role for Dll4-Notch signaling in regulating Treg development in EAE, making it an encouraging target for Treg-mediated immunotherapy in autoimmune diseases, such as multiple sclerosis.