The T cell leukemia 3 (Tlx3) gene has been implicated in specification of glutamatergic sensory neurons in the spinal cord. In cranial sensory ganglia, Tlx3 is highly expressed in differentiating neurons during early embryogenesis. To study a role of Tlx3 during neural differentiation, mouse embryonic stem (ES) cells were transfected with a Tlx3 expression vector. ES cells stably expressing Tlx3 were grown in the presence or absence of a neural induction medium. In undifferentiated ES cells, there was no significant difference in gene expression in the presence or absence of Tlx3, even after ES cells were cultured for an extensive time period. In contrast, expression levels of Mash1, Ngn1, and NeuroD were significantly higher in Tlx3-expressing cells after neural induction for 4 days compared with those in cells expressing the control vector. At 7 days after neural induction, whereas expression of the proneural genes was down-regulated, VGLUT2, GluR2, and GluR4 were significantly increased in ES cell-derived neurons expressing Tlx3. The sequential and coordinated expression of the proneural and neuronal subtype-specific genes identifies Tlx3 as a selector gene in ES cells undergoing neural differentiation. In addition, the differential effects of Tlx3 overexpression in undifferentiated ES cells compared with ES cell-derived neurons suggest that Tlx3 exerts context-dependent transcriptional signals on its downstream target genes. The context-dependent function of Tlx3 as a selector gene may be used to establish a novel strategy to conditionally generate excitatory glutamatergic neurons from ES cells to cure various types of neurodegenerative disorders.