P19 cells, a line of pluripotent embryonal carcinoma derived from C3H/He mice, can differentiate into neuronal cell morphology. It is already known that neuronally differentiated P19 cells mainly produce gamma-amino butyric acid (GABA) as a neurotransmitter. In the present study we investigated GABAergic neuron-specific and neuronal differentiation markers, as indexes of the acquisition processes of neuronal phenotype, and explored the possibility for an in vitro model of brain development during perinatal period. Subconfluent P19 cells cultured in DMEM containing 10% FBS were harvested and seeded to form small aggregates (embryoid body: EB) in DMEM with 2.5% FBS and retinoic acid. At 4 days after the induction of EB, the aggregates were dispersed by trypsin and replated in DMEM containing N-2 and B27 supplements. The cells were then harvested at 0, 6, and 9 days after the induction of neuronal differentiation (ND), and cDNAs in each time period were obtained. The gene expression of markers to evaluate the stage of neuronal differentiation, GABA synthases, and GABA transporter were determined using RT-qPCR. DCX, a marker of the stage from neuroblast to immature neurons, was extremely low expression level at EB, and reached to the higher levels at 6 days of ND. NeuN, a marker of mature neurons, was also not detected during EB, and linearly elevated until 9 days after the induction of ND. The gene expression of GAD1, encoded a GABA synthase protein, showed already higher level at 6 days after ND and maintained the level to 9 days. The expression level of GAD2 gene, encoded another GABA synthase protein, was approximately 25% at day 6 and 50% at day 9 after ND to the level of GAD1. GAT-1, GABA transporter gene, was rapidly elevated at day 6 after ND and stayed the level at day 9. These results suggest that the synthesis of GABA as a neurotransmitter might be manifested in P19 cells with mature neuronal phenotype, but cytoplasmic GABA, non-neurotransmitter, starts to synthesize in the cells during the immature neuronal stage. Furthermore, these profiles of gene expression in P19 cells were compared with those of cerebral cortex in mice during pre- and postnatal periods.