The vertebrate neural crest is a population of migratory cells that originates in the dorsal aspect of the embryonic neural tube. These cells undergo an epithelial-to-mesenchymal transition (EMT), delaminate from the neural tube and migrate extensively to generate an array of differentiated cell types. Elucidating the gene regulatory networks involved in neural crest cell induction, migration and differentiation are thus crucial to understanding vertebrate development. To this end, we have identified Annexin A6 as an important regulator of chick midbrain neural crest cell emigration. Annexin proteins comprise a family of calcium-dependent, membrane-binding molecules that mediate a variety of cellular and physiological processes including cell adhesion, migration and invasion. Our data indicate that Annexin A6 is expressed in the proper spatio-temporal pattern in the chick midbrain to play a potential role in neural crest cell ontogeny. To investigate Annexin A6 function, we have depleted or overexpressed Annexin A6 in the developing midbrain neural crest cell population. Our results show that knock-down or overexpression of Annexin A6 reduces or expands the migratory neural crest cell domain, respectively. Importantly, this phenotype is not due to any change in cell proliferation or cell death but can be correlated with changes in the size of the premigratory neural crest cell population and with markers associated with EMT. Taken together, our data indicate that Annexin A6 plays a pivotal role in modulating the formation of cranial migratory neural crest cells during vertebrate development.