Rathke's pouch contains progenitor cells that differentiate into the endocrine cells of the pituitary gland. It gives rise to gonadotrope, thyrotrope, somatotrope, corticotrope and lactotrope cells in the anterior lobe and the intermediate lobe melanotropes. Pituitary precursor cells express many members of the Notch signaling pathway including the downstream effector gene Hes1. We hypothesized that Hes1 regulates the timing of precursor differentiation and cell fate determination. To test this idea, we expressed Hes1 in differentiating pituitary cells and found that it can inhibit gonadotrope and thyrotrope differentiation. Pituitaries of Hes1 deficient mice have anterior lobe hypoplasia. All cells in the anterior lobe are specified and differentiate, but an early period of increased cell death and reduced proliferation causes reduced growth, evident as early as e14.5. In addition, cells within the intermediate lobe differentiate into somatotropes instead of melanotropes. Thus, the Hes1 repressor is essential for melanotrope specification. These results demonstrate that Notch signaling plays multiple roles in pituitary development, influencing precursor number, organ size, cell differentiation and ultimately cell fate.