The A13 dopaminergic nucleus belongs to the incerto-hypothalamic area. It is thought to exert autonomous roles by integrating sensory input to autonomic, neuroendocrine, and motor output. Although its early development has been well characterized, the factors that contribute to later steps of its formation remain unknown. Transcription factors of the Onecut family have been detected in the A13 nucleus, raising the question of possible roles of these factors during A13 development. Using a combination of immunofluorescence analyses on sections and after whole-mount labeling followed by 3D reconstructions, we further characterized the second phase of development of the A13 nucleus in the mouse, described the distribution of the Onecut proteins throughout A13 development, and analyzed the phenotype of this nucleus in single or compound mutant embryos for the Onecut factors. Here we show that A13 development can be divided into two successive phases. First, during radial migration toward the pial surface the A13 cells differentiate into dopaminergic neurons. Second, these cells gather in the vicinity of the third ventricle. Onecut factors are dynamically and differentially expressed in the A13 nucleus during these two phases of development. In Onecut mutant embryos, the A13 neurons differentiate normally but scatter in the diencephalon and fail to properly gather close to the third ventricle. Hence, Onecut factors are markers of the A13 nucleus throughout embryonic development. They are dispensable for the first phase of A13 development but are required for the second phase of development and for maintenance of this nucleus. J. Comp. Neurol. 520:1424–1441, 2012. © 2011 Wiley Periodicals, Inc.