DAX1 encoded by NR0B1, when mutated, is responsible for X-linked adrenal hypoplasia congenita (AHC). AHC is due to failure of the adrenal cortex to develop normally and is fatal if untreated. When duplicated, this gene is associated with an XY sex-reversed phenotype. DAX1 expression is present during development of the steroidogenic hypothalamic-pituitary-adrenal-gonadal (HPAG) axis and persists into adult life. Despite recognition of the crucial role for DAX1, its function remains largely undefined. The phenotypes of patients and animal models are complex and not always in agreement. Investigations using cell lines have proved difficult to interpret, possibly reflecting cell line choices and their limited characterization. We will review the efforts of our group and others to identify appropriate cell lines for optimizing ex vivo analysis of NR0B1 function throughout development. We will examine the role of DAX1 and its network partners in development of the hypothalamic-pituitary-adrenal/gonadal axis (HPAG) using a variety of different types of investigations, including those in model organisms. This network analysis will help us to understand normal and abnormal development of the HPAG. In addition, these studies permit identification of candidate genes for human inborn errors of HPAG development.