High-level γ-globin gene expression for definitive therapy of beta globin disorders likely requires γ-globin gene promoter activation, dissociation of repressor complexes, and/or local chromatin modification. To define the molecular mechanisms employed by high-potency γ-globin inducers, the effects of butyrate on transcriptional co-regulatory protein recruitment to the human γ- and β-globin gene promoters were defined and compared to those of novel SCFAD γ-globin inducers recently identified through computational modeling. One particularly potent SCFAD inducer, RB7, which has no inhibitory effect on class I HDAC enzymes (HDAC1, HDAC2, and HDAC3), demonstrated 5-fold induction, (2.5 to 3-fold greater γ-globin gene-inducing activity than butyrate), in a transcriptional reporter assay which detects only strong γ-globin gene inducers. Chromatin immunoprecipitation (ChIP) assays performed on the integrated human gamma and beta globin gene promoters demonstrated that RB7, butyrate, and other transcriptionally-active SCFADs, induced dissociation of HDAC-3, and its adaptor protein NCoR, from the γ-globin gene promoter, with no such effect on the β-globin promoter, coincident with recruitment of RNA Polymerase II to the γ-globin promoter and initiation of γ-globin transcription. In a reciprocal fashion, as HDAC-3 dissociated, the chromatin-modifying ATPases Brg-1 and Brm were recruited specifically to the γ-globin gene promoter in response to the active SCFADs. The ability of the novel SCFADs to induce dissociation of HDAC-3 (but not HDAC-1 or -2) from the γ-globin promoter was proportional to the degree of γ-globin promoter induction by these agents. Knockdown of HDAC-3 by siRNA induced transcription of γ-globin specifically, demonstrating the functional importance of the activity of SCFADS in dissociating HDAC-3 from the γ-globin gene promoter. These studies demonstrate new dynamic alterations in transcriptional regulatory complexes specifically-associated with high-level activation of γ-globin and also identify essential molecular targets for future therapeutic interventions.