Abstract Pituitary gland dysfunction affects growth, fertility, the stress response, and many other physiological functions. Hypopituitarism can result from congenital defects in organ development (about 1/4000 births) and from pituitary adenomas, which are among the most common type of intracranial tumor. Treatment involves hormone replacement therapy, which can involve daily injections of recombinant growth hormone at great expense per patient. Some adenomas respond to pharmacological therapy, while others are recurrent, potentially causing disfigurement, multiple trans-sphenoidal surgeries, and can result in blindness and death. Genetically engineered mice have been used to identify the roles of several transcription factors and signaling molecules in differentiation, and there is excellent correspondence to human pituitary disorders. In previous years of this grant we used mouse mutants to define the roles of several transcription factors (PITX2, GATA2, FOXL2, LHX3, LHX4 and ISL1) and signaling pathways (BMP, FGF, WNT) in pituitary development and disease. We also determined how the transcription factor mutations affected expression of cell cycle regulators to cause pituitary hypoplasia, and identified a novel gene that drives cell proliferation in a mouse model of thyrotrope adenoma. The long-term goal of this research is to improve diagnosis and therapy for people with genetic or acquired pituitary disease by increasing our fundamental understanding of how pituitary growth and cell specification are orchestrated at the molecular level. The overall objectives of this application are: to define the roles of two key transcription factors, Isl1 and Zeb2, in pituitary development and disease, and to employ an unbiased approach to identify novel transcription factors, lncRNA and chromatin changes that drive pituitary thyrotrope cell fate and establishment of robust hormone production. We generated the first model of Rathke's cleft cysts by deleting Isl1 in pituitary development, and we have evidence that this leads to misregulation of pituitary stem cells and formation of adenomas. We also show that Zeb2 is necessary to drive an epithelial to mesenchymal-like transition (EMT) in pituitary stem cell cultures. The proposed research uses up to date approaches for analysis of Isl1 and Zeb2. It is innovative because it uses an unbiased approach to discovering new factors and pathways, including exploring the role of noncoding RNAs, which is a relatively new area of investigation, and it uses state of the art genomic analysis techniques to identify regulatory regions of genes, which are likely sites of mutations in patients with hypopituitarism or adenoma risk factors. This research will be significant because it will provide fundamental information about the molecular mechanisms of formation of pituitary cysts and adenomas, the regulation of EMT, and it will establish the regulatory mechanisms underlying thyrotrope fate. This information will contribute to identification of the basis for human pituitary diseases including congenital hormone deficiencies and adenomas.