The neural crest is a unique structure in vertebrates. Neural crest cells play important roles in the formation of organs that characterize the vertebrate body plan. In this unit, we describe a primary culture method for mouse mesencephalic neural crest cells. The neural crest cells cultured by this method actively proliferate and differentiate into various cell types that originate from cranial neural crest cells, such as chondrocytes, neurons, and glia. Therefore, this primary culture method is useful for analyzing the development of mouse mesencephalic neural crest cells.