The tumor suppressor gene p53 is a transcription factor that mediates both cell cycle arrest and apoptosis. Interestingly, p53 also induces differentiation of a number of tissues, including leukemic cells. However, although p53-mediated differentiation of leukemic U-937 cells depends on the transcriptional activity of p53, a p53 target gene mediating differentiation has hitherto not been identified. To screen for novel p53 target genes in leukemic cells, a cDNA microarray analysis was performed with U-937-4/ptsp53 cells, expressing a temperature-sensitive p53 mutant. We report that transcription of the Staf50 (stimulated transacting factor of 50 kDa) gene is upregulated in response to wild-type p53 in U-937-4, K562 and MCF-7 cells. Staf50 was directly activated by p53, as determined by the independence of de novo protein synthesis. Moreover, while the proximal promoter of Staf50 was found not to be p53 responsive, a functional enhancer-like p53-response element in intron 1 of the Staf50 gene was identified that was also transactivated by the p53-family member p73. Direct binding of p53 to the response element was shown by electrophoretic mobility shift analysis. Ectopic expression of Staf50 in U-937 cells resulted in reduced clonogenic growth. Moreover, levels of endogenous Staf50 mRNA correlated to all-trans retinoic acid-induced differentiation of promyelocytic NB-4 and HL60 cells, suggesting that Staf50 could be involved in proliferation and/or differentiation of leukemic cells.