During development of the zebrafish embryo, glycine signaling promotes the differentiation of neural stem cells (NSCs). We found that glycine signaling suppresses the expression of Ligand of Numb X1 (lnx1, Ligand of numb protein-x1), a gene of unknown function during NSC differentiation that is selectively expressed in the embryonic central nervous system (CNS). As a consequence, Numb levels were stabilized and Notch activity (measured as her4.1 expression) was reduced, promoting NSC differentiation. These consequent actions were blocked by knockdown of lnx1. In contrast, lnx1 overexpression increased NSC proliferation and led to defects of neural tube closure at the early stages of development. Thus, our data provide evidence that glycine/lnx1 signaling modulates NSC proliferation by regulation of Notch signaling.