Overexpression of the transcription factor (TF) ATOH1 is known to induce the transformation of nonsensory cells in the organ of Corti into hair cells (HCs). Evaluating DNA 5Πto the coding sequence of the pou4f3 gene, a target of ATOH1 in HCs, we identified in three regions containing clustered binding sites for ATOH1 and several other TFs that are expressed in developing inner ear sensory epithelia at the time of HC specification. These regions and sites are highly conserved across evolutionarily distant mammalian species. To test the hypothesis that the identified TFs act in combination to regulate the pou4f3 gene, we transfected by electroporation neonatal cochlear sensory epithelium from mice expressing green fluorescent protein (GFP) under the control of an 8.5-kb 5' pou4f3 genomic fragment. Plasmids encoding 21 TFs c-transfected with human ATOH1 (hATOH1). Cotransfection with hETV4, hNMYC, or hETS2 produced significantly more pou4f3/GFP and myosin 7A-positive nonsensory cells than hATOH1 alone. Co-transfection of hATOH1 with hHES1, hHES5, or hNEUROD1 reduced the effects of hATOH1. Chromatin immunoprecipitation (ChIP)of DNA from an inner ear cell line transfected with hNMYC,hETV4, or hETS2 revealed binding to a conserved region immediately proximal to the coding sequence. ChIP similarly revealed binding of hGATA3, hNMYC, and hTFE2 to a region several kilobases distal to the coding sequence, which we have previously shown to bind ATOH1. The results suggest that ATOH1 acts in concert with a subset of other TFs to directly regulate the pou4f3 gene and more broadly to regulate the HC phenotype.