Relatively little is known about the development of the thalamus, especially its differentiation into distinct nuclei. We demonstrate here that Gbx2-expressing cells in mouse diencephalon contribute to the entire thalamic nuclear complex. However, the neuronal precursors for different thalamic nuclei display temporally distinct Gbx2 expression patterns. Gbx2-expressing cells and their descendents form sharp lineage-restriction boundaries delineating the thalamus from the pretectum, epithalamus and prethalamus, revealing multiple compartmental boundaries within the mouse diencephalon. Without Gbx2, cells originating from the thalamus abnormally contribute to the epithalamus and pretectum. This abnormality does not result from an overt defect in patterning or cell-fate specification in Gbx2 mutants. Chimeric and genetic mosaic analysis demonstrate that Gbx2 plays a cell-nonautonomous role in controlling segregation of postmitotic thalamic neurons from the neighboring brain structures that do not express Gbx2. We propose that, within the developing thalamus, the dynamic and differential expression of Gbx2 may be involved in the specific segregation of thalamic neurons, leading to partition of the thalamus into different nuclei.