Background: The neurons contributing to thalamic nuclei are derived from at least two distinct progenitor domains: the caudal (cTH) and rostral (rTH) populations of thalamic progenitors. These neural compartments exhibit unique neurogenic patterns, and the molecular mechanisms underlying the acquisition of neurotransmitter identity remain largely unclear. Results: T-cell acute lymphocytic leukemia protein 1 (Tal1) was expressed in the early postmitotic cells in the rTH domain, and its expression was maintained in mature thalamic neurons in the ventrolateral geniculate nucleus (vLG) and the intergeniculate leaflet (IGL). To investigate a role of Tal1 in thalamic development, we used a newly generated mouse line driving Cre-mediated recombination in the rTH domain. Conditional deletion of Tal1 did not alter regional patterning in the developing diencephalon. However, in the absence of Tal1, rTH-derived thalamic neurons failed to maintain their postmitotic neuronal features, including neurotransmitter profile. Tal1-deficient thalamic neurons lost their GABAergic markers such as Gad1, Npy, and Penk in IGL/vLG. These defects may be associated at least in part with down-regulation of Nkx2.2, which is known as a critical regulator of rTH-derived GABAergic neurons. Conclusions: Our results demonstrate that Tal1 plays an essential role in regulating neurotransmitter phenotype in the developing thalamic nuclei. Developmental Dynamics 246:749–758, 2017.