The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules)despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a γ-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by aγ-secretase inhibitor. These results establish distinct (non-redundant),instructive roles for Notch receptors in nephron segmentation.