Cells destined to become the neurones of the cochlear-vestibular ganglion (CVG) originate within the otic epithelium. Early in development they detach from their neighbors and migrate out of the epithelium, where they coalesce to form the CVG. To accomplish this process, the neuroblasts must modify their interactions with other cells within the epithelium and with proteins in the extracellular matrix to allow for repositioning. The aim of this study was to investigate the roles of the major families of adhesion molecules that mediate cellular interactions with the extracellular matrix, the integrins, and with other cells, the cadherins, in neuroblast segregation from the otic epithelium. The expression of classical cadherins increased in migrating neuroblasts compared with the otic epithelium. Quantitative RT-PCR revealed that this was concomitant with down-regulation of E-cadherin and up-regulation of N-cadherin in the migrating cells. In contrast, the level of β1 integrin expression by the epithelium was maintained in migrating neuroblasts. However, although multiple integrin ligands were expressed within the otic basement membrane at this stage of development, only fibronectin (FN) supported neuroblast migration along the substrate in vitro. Inhibition of β1 integrins resulted in significantly reduced linear migration on FN. Importantly, neuroblasts retained the ability to segregate from the epithelium but remained compacted immediately adjacent to the originating tissue, suggesting dominance of cell–cell over cell–matrix interactions. These data suggest that the balance between cell–cell and cell–substratum interactions directs otic neuroblast migration and gangliogenesis. © 2011 Wiley-Liss, Inc.