The neural tube comprises several different types of progenitors and postmitotic neurons that coordinately act with each other to play integrated functions. Its development consists of two phases: proliferation of progenitor cells and differentiation into postmitotic neurons. How progenitor cells differentiate into each corresponding neuron is an important question for understanding the mechanisms of neuronal development. Here we introduce one of the Sox transcription factors, Sox14, which plays an essential role in the promotion of neuronal differentiation. Sox14 belongs to the SoxB subclass and its expression starts in the progenitor regions before neuronal differentiation is initiated at the trunk level of the neural tube. After neuronal differentiation is initiated, Sox14 expression gradually becomes confined to the V2a region of the neural tube, where Chx10 is co-expressed. Overexpression of Sox14 restricts progenitor cell proliferation. Conversely, the blockade of Sox14 expression by the RNAi strategy inhibits V2a neuron differentiation and causes expansion of the progenitor domain. We further found that Sox14 acted as a transcriptional activator. Taken together, Sox14 acts as a modulator of cell proliferation and an initiator protein for neuronal differentiation in the intermediate region of the neural tube.