Enhancers have been functionally described for >35 years, but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood. Using a combination ofin vivoenhancer mapping and proteomics approaches, we have established that two distant and distinct early enhancers, each requiring different transcription complexes, are required for full activation of the gene encoding the pituitary lineage determining factor, Pit1. A transcription factor belonging to the “giant, multiple-homeodomain and zinc finger family,” Atbf1, serves as a novel pituitary regulator for one of the two required enhancers as shown by genetic andin vitroanalysis.