The six-layer structure is a unique feature of the mammalian neocortex, where each layer is composed of specific subtypes of glutamatergic projection neurons. The production sequence of these layer subtypes follows a temporal program, which is conserved among mammalian species. Although increased number of genes have been identified that mediate gene regulatory network between the layer neurons, how the sequential production of layer subtypes are instructed and the mechanism by which layer subtypes diverge during development remain unclear. The forkhead family transcription factor Foxg1 is a critical regulator of early cortical program and also plays important roles in upper-layer neurogenesis. To assess the mechanisms by which Foxg1 regulates corticogenesis program, we designed a doxycycline-inducible system to manipulate the expression of Foxg1 at specific developmental stages, and identified target genes of Foxg1 required for the specification of upper-layer neuron subtypes. Among these, a neuron-specific RNA binding protein Nova1 showed a significant up-regulated response in gene expression to Foxg1 inactivation in vivo. We further identified a novel isoform of Nova1 that is expressed within cortical ventricular zone and postmitotic neurons, whereas its full-length conterparts was predominantly expressed in subpopulations of layer 6 and subplate neurons, indicating differential requirement within neocortical neuron subtypes. Comparative analysis revealed that the novel isoform of Nova1 is specific to the mammalian vertebrates, suggesting that expression of Nova1 isoforms in the mammalian brain may play important roles in the development of the neocortex.