Stem cells are a potential key strategy for treating neurodegenerative diseases in which the generation of new neurons is critical. A better understanding of the characteristics and molecular properties of neural stem cells (NSCs) and differentiated neurons can help with assessing neuronal maturity and, possibly, in devising better therapeutic strategies. We have performed an in-depth gene expression profiling study of murine NSCs and primary neurons derived from embryonic mouse brains. Microarray analysis revealed a neuron-specific gene expression signature that distinguishes primary neurons from NSCs, with elevated levels of transcripts involved in neuronal functions, such as neurite development and axon guidance in primary neurons and decreased levels of multiple cytokine transcripts. Among the differentially expressed genes, we found a statistically significant enrichment of genes in the ephrin, neurotrophin, CDK5, and actin pathways, which control multiple neuronal-specific functions. We then artificially blocked the cell cycle of NSCs with mitomycin C (MMC) and examined cellular morphology and gene expression signatures. Although these MMC-treated NSCs displayed a neuronal morphology and expressed some neuronal differentiation marker genes, their gene expression patterns were very different from primary neurons. We conclude that 1) fully differentiated mouse primary neurons display a specific neuronal gene expression signature; 2) cell cycle block at the S phase in NSCs with MMC does not induce the formation of fully differentiated neurons; 3) cytokines change their expression pattern during differentiation of NSCs into neurons; and 4) signaling pathways of ephrin, neurotrophin, CDK5, and actin, related to major neuronal features, are dynamically enriched in genes showing changes in expression level.