Analysis of an allelic series in mice revealed that the Pitx2 homeobox gene is required at multiple stages of pituitary development. It is necessary for initiating expansion of Rathke's pouch and maintaining expression of the fetal-specific transcription factors Hesx1 and Prop1. At later stages Pitx2 is necessary for specification and expansion of the gonadotropes and Pit1 lineage within the ventral and caudomedial anterior pituitary. Mechanistically, this is due to the dependence of several critical lineage-specific transcription factors, Pit1, Gata2, Egr1 and Sf1, on a threshold level of PITX2. The related Pitx1 gene has a role in hormone gene transcription, and it is important late in ontogeny for the final expansion of the differentiated cell types. Pitx1 and Pitx2 have overlapping functions in the expansion of Rathke's pouch, revealing the sensitivity of pituitary organogenesis to the dosage of the PITX family. The model developed for PITX gene function in pituitary development provides a better understanding of the etiology of Rieger syndrome and may extend to other PITX-sensitive developmental processes.