Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases.