FOXL2 is a forkhead transcription factor expressed in the eye, ovary and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 co-localizes with the glycoprotein hormone -subunit (GSU) in quiescent cells of the mouse pituitary from e11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and GSU suggested that the GSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (T3-1, LT2), thyrotrope-derived (thyrotropin) and heterologous (CV-1) cells in a context dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.