AbstractTo understand the molecular basis of the specification of thalamic nuclei, we analyzed the expression patterns of various transcription factors and defined progenitor cell populations in the embryonic mouse thalamus. We show that the basic helix‐loop‐helix (bHLH) transcription factor Olig3 is expressed in the entire thalamic ventricular zone and the zona limitans intrathalamica (ZLI). Next, we define two distinct progenitor domains within the thalamus, which we name pTH‐R and pTH‐C, located caudal to the ZLI. pTH‐R is immediately caudal to the ZLI and expresses Nkx2.2, Mash1, and Olig3. pTH‐C is caudal to pTH‐R and expresses Ngn1, Ngn2, and Olig3. Short‐term lineage analysis of Olig3‐, Mash1‐, Ngn1‐, and Ngn2‐expressing progenitor cells as well as tracing the Pitx2 cell lineage suggests that pTH‐C is the only major source of thalamic nuclei containing neurons that project to the cerebral cortex, whereas pTH‐R and ZLI are likely to produce distinct postmitotic populations outside of the cortex‐projecting part of the thalamus. To determine if pTH‐C is composed of subdomains, we characterized expression of the homeodomain protein Dbx1 and the bHLH protein Olig2. We show that Dbx1 is expressed in caudodorsal‐high to rostroventral‐low gradient within pTH‐C. Analysis of heterozygous Dbx1nlslacZ knockin mice demonstrated that Dbx1‐expressing progenitors preferentially give rise to caudodorsal thalamic nuclei. Olig2 is expressed in an opposite gradient within pTH‐C to that of Dbx1. These results establish the molecular heterogeneity within the progenitor cells of the thalamus, and suggest that such heterogeneity contributes to the specification of thalamic nuclei. J. Comp. Neurol. 505:73–91, 2007. © 2007 Wiley‐Liss, Inc.