T cell maturation into TCRalphabeta(+) or TCRgammadelta(+) cells from common immature CD4(-)CD8(-)(DN) precursors occurs in the thymus, and is controlled through ordered regulation of genes. The aryl hydrocarbon receptor (AHR), a latent cytoplasmic transcription factor, affects thymocyte maturation and differentiation at several stages, also including DN cells. We analyzed in murine fetal thymus organ cultures (FTOC) the outcome of AHR-signaling and found a higher frequency of DN TCRgammadelta(+) cells in the presence of the AHR-activating ligand TCDD. We detected a novel population of CD25(int/lo)CD44(hi) cells associated with preferential emigration and a TCRgammadelta(+) T cell fate of thymocytes. Sorted DN TCRgammadelta(+) emigrants could proliferate if IL-2 was available. Moreover, they suppressed the proliferation of co-cultivated, activated CD4(+) T cells. Gene expression profiles of purified DN emigrants from TCDD*FTOC revealed 295 modulated genes, 10% of which are genes of the immune system. For instance, RAG-1, TdT, and Gfi-1 were downregulated, yet genes indicative of mature thymocytes were upregulated. In conclusion, we have detected changes in the differentiation programme of fetal DN thymocytes after ligand-activation of the AHR. In particular, we observed a higher frequency of DN TCRgammadelta(+) cells with high emigration potential, and possible regulatory functions.