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Transcriptional silencing via promoter methylation of genes important for cell growth and differentiation plays a key role in myeloid leukemogenesis. We find that clinically achievable levels of 5-aza-2¶-deoxycytidine (5-AZA-dC), a potent inhibitor of DNA methylation, can modify chromatin and restore the ability of tumor necrosis factor A (TNFA) to induce monocytic differentiation of the acute myeloid leukemia cells NB4 and U937. Although 5-AZA-dC cannot fully induce differentiation, we show that 5-AZA-dC acts directly on TNFA-responsive promoters to facilitate TNFA-induced transcriptional path-ways leading to differentiation. 5-AZA-dC regulates the expression of Dif-2, a TNFA target gene, by deacetylating chromatin domains in a methylation-dependent manner. Chromatin immunoprecipitation analyses of the Dif-2 pro-moter show histone hyperacetylation and a recruitment of the nuclear factor-KB transcription factor in response to 5-AZA-dC. Furthermore, 5-AZA-dC plus TNFA enhances the level of phosphorylated RNA Pol II at the Dif-2 promoter via synergistic recruitment of TFIIH. We conclude that nonspe-cific changes in chromatin can allow a specific transcriptional inducer to overcome blocks in leukemic cell differentiation. Our results support the concept of low doses of 5-AZA-dC acting in combination with other agents to target epigenetic changes that drive malignant growth in leukemic cells. [Cancer Res 2009;69(1):55–64