Based on a knocked-out mouse model and a few expression studies, TLX3 is regarded as a homeobox gene crucial for the development of the autonomic nervous system. This gene can undergo rearrangements or deregulation, giving rise to T-cell acute lymphocytic leukemia. The present report is focused on the identification of elements and factors playing a role in the TLX3 physiologic expression regulation and therefore likely to be involved in cancer development. In particular, after identifying the transcription start points, we have made use of in vitro transfection assays to show that the 5'-untranslated region of the gene is necessary for the basal promoter activity in cell lines from different origin. By site-directed mutagenesis, two tandem CCAAT boxes have been localized as critical elements of this region. In vivo chromatin immunoprecipitation and electrophoretic mobility shift assays have indicated that nuclear factor Y (NFY) recognizes these sites in all the analyzed cell lines. The physiologic role of such an interaction has been confirmed by a dominant-negative version of the NFY transcription factor that has turned out to decrease both in vitro TLX3 promoter activity and endogenous amount of mRNA. Finally, a consistent in vivo TLX3 expression impairment was also achieved after NFY mRNA knockdown. The full characterization of the TLX3 transcription regulation will ultimately provide crucial elements to define the involvement of this gene in T-cell acute lymphocytic leukemia development.