IFN-stimulated gene (ISG) expression is mediated by the ISGF3 transcription factor complex, composed of tyrosine phosphorylated STAT1 and STAT2 and the DNA binding partner, IRF9. The transactivation domain of STAT2 recruits transcriptional activators and chromatin modifiers that regulate polymerase recruitment and elongation. Interestingly, both histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities are required for transcription of ISGs and establishment of an antiviral state. Inhibition of HDAC activity or reduction of HDAC1, 2, and 3 abrogates ISG transcription without altering the activation or chromatin recruitment of ISGF3. To pinpoint this HDAC requirement, we examined ISG transcription in vitro and ISG epigenetic regulation in vivo. While transcription of ISGs on nucleosome-free DNA in vitro was unaffected by HDAC activity, chromosomal remodeling occurred at ISG promoters in vivo in response to IFN stimulation and this remodeling required HDAC activity. To discover factors required for ISG transcription, we purified native ISGF3 complexes and identified STAT2-interacting proteins by mass spectrometry. The DNA helicases Rvb1 and Rvb2 associated with the transactivation domain of STAT2 and reducing their expression by RNA interference impaired ISG transcription. Neither Rvb1 nor Rvb2 were required for induction of IFN-gamma or TNF-alpha induced transcription. Interestingly, IFN-alpha stimulation recruited RNA Pol II to ISG promoters even in the absence of HDAC activity, and the recruited Pol II became phosphorylated on Ser-5, a hallmark of transcriptional initiation. However, RNA Pol II did not become phosphorylated on Ser-2 and failed to transcribe IFN target genes in the absence of HDAC activity. Moreover, BRD proteins, PAF1, and SAGA complexes were found to regulate ISG transcription, including requirements for regulated and possibly sequential histone acetylation, deacetylation, and ubiquitinylation. Individual chromatin modification steps were impaired when HAT or HDAC enzymes were inhibited. These data define an ordered set of chromatin modifications that coordinate individual regulatory events necessary for recruitment of positive factors and dismissal of negative factors during the ISG transcriptional initiation and elongation cycle.