A variety of transcription factors including Wt-1, Sf-1, Dax-1 and Pbx1 have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1^+/-) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline Pcna levels are lower in Pbx1^+/- mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of insulin-like growth factors in adrenocortical proliferation and development, real time RT-PCR demonstrates significant lower expression levels of the Igf1 receptor, and up-regulation of Igfbp2. Functionally, Pbx1^+/- mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.