Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although recent work has shown that receptor recycling plays an important role in NCC motility on laminin, the molecular mechanisms regulating NCC motility on fibronectin remain unclear. One mechanism by which cells regulate motility is by modulating the affinity of integrin receptors. Here, we provide evidence that cranial and trunk NCCs rely on functional regulation of integrins to migrate efficiently on fibronectin (FN) in vitro. For NCCs cultured on fibronectin, velocity decreases after Mn2+ application (a treatment that activates all surface integrins) while velocity on laminin (LM) is not affected. The distribution of activated integrin beta 1 receptors on the surface of NCCs is also substratum-dependent. Integrin activation affects cranial and trunk NCCs differently when cultured on different concentrations of FN substrata; only cranial NCCs slow in a FN concentration-dependent manner. Furthermore, Mn2+ treatment alters the distribution and number of activated integrin beta 1 receptors on the surface of cranial and trunk NCCs in different ways. We provide a hypothesis whereby a combination of activated surface integrin levels and the degree to which those receptors are clustered determines NCC motility on fibronectin.