The proneural basic helix-loop-helix proteins play a crucial role in promoting the differentiation of postmitotic neurons from neural precursors. However, recent evidence from flies and frogs indicates that additional factors act together with the proneural bHLH proteins to promote neurogenesis. We have identified a novel zinc finger protein, neuronal Kruppel-like protein (NKL), that positively regulates neurogenesis in vertebrates. NKL is expressed in Xenopus primary neurons and in differentiating neuronal precursors in the intermediate zone of the mouse and chick neural tube. In frog embryos, NKL is induced by overexpression of Neurogenin (Ngn), arguing that NKL is downstream of the proneural determination genes. Our results show that NKL and a NKL/VP16 fusion protein promote differentiation of neuronal precursors in the embryonic chick spinal cord. Following in ovo misexpression of NKL, neuroepithelial cells exit the cell cycle and differentiate into neurons. Similarly, NKL/VP16 induces extra primary neurons in frogs and upregulates expression of the neural differentiation factors, Xath3 and MyT1, as well as the neuronal markers, N-tubulin and elrC. Our findings establish NKL as a novel positive regulator of neuronal differentiation and provide further evidence that non-bHLH transcription factors function in the neuronal differentiation pathway activated by the vertebrate neuronal determination genes.