It is important for neural crest cells, especially trunk crest, to have sufficient cell-free extracellular space so they can migrate normally. Neural crest cells exert relatively weak traction forces on the substrate, an attribute that allows them to migrate on or through relatively weak extracellular matrices. Hyaluronan (hyaluronic acid) is involved in the initial separation of neural crest cells from the neural tube and opening up of spaces through which they migrate. Neural crest cells produce proteases such as plasminogen activator that help create the spaces through which they migrate.

Cranial and trunk cells use different mechanisms of attachment to extracellular matrices. Trunk neural crest cells attach to fibronectin, laminin and collagen types I and IV. Cranial neural crest cells do not attach to the two collagens although they do attach to basal laminae. Attachment of trunk neural crest cells to fibronectin is calcium-dependent; attachment of cranial crest cells is not. Attachment to and migration over these substrate molecules is mediated by the family of attachment proteins called integrins. Organized basal laminae (e.g. those of the surface ectoderm and ventral neural tube) act as barriers that guide migrating crest cells along their surfaces. Other extracellular matrix molecules, notably chondroitin sulfate-rich proteoglycans, are not good substrates for migrating crest cells. As a result, few neural crest cells are seen in areas that contain high concentrations of chondroitin sulfate such as the sclerotome of the somites. This principle is well-illustrated by the distribution of migrating neural crest cells in the somites; neural crest cells enter only the anterior parts of the somites, where many of them settle to form the sensory ganglia. They do not penetrate the posterior regions of the somites, which have elevated concentrations of chondroitin sulfate.

From studies and experiments of transplantation, it is known that directionality of migration is not an intrinsic property of neural crest cells. For example, neural crest cells that are grafted to a different part of the embryo do not seek out their original pathway, but instead pick up the pathway that is characteristic of that area. See: Migration Experiments

Extracellular matrices play a significant role in the migration of neural crest cells. Neural crest cells use molecular cues from their environment to migrate. This has been observed in experiments in which neural crest cells were cultured on or in various extracellular matrices. The extracellular matrices is composed of a meshwork of fibers that undergoes structural alterations and biochemical changes when neural crest cells migrate in or on it. The matrix can contain proteoglycans, collagens, chondroitin sulfate (as mentioned earlier it prevents migration), fibronectin, and laminin.