The T-box transcription factor Tpit was identified as a cell-specific factor for expression of the pituitary proopiomelanocortin (POMC) gene. Expression of this factor is exclusively restricted to the pituitary POMC-expressing lineages, the corticotrophs and melanotrophs. We have now determined the role of this factor in pituitary cell differentiation. Tpit is a positive regulator for late POMC cell differentiation and POMC expression, but it is not essential for lineage commitment. The pituitary intermediate lobe normally contains only Tpit-expressing melanotrophs. Inactivation of the Tpit gene results in almost complete loss of POMC-expressing cells in this tissue, which now has a large number of gonadotrophs and a few clusters of Pit-1-independent thyrotrophs. The role of Tpit as a negative regulator of gonadotroph differentiation was confirmed in transgenic gain-of-function experiments. One mechanism to account for the negative role of Tpit in differentiation may be trans-repression between Tpit and the gonadotroph-restricted factor SF1. These data suggest that antagonism between Tpit and SF1 may play a role in establishment of POMC and gonadotroph lineages and that these lineages may arise from common precursors.