During eye formation, periocular neural crest cells (pNC) migrate and differentiate to form the anterior ocular structures. In the chick cornea, this involves two waves of migration that result in the formation of the corneal endothelium and stroma. Abnormalities in pNC migration lead to corneal malformation, such as anterior segment dystrophy. Corneal dystrophies, infections, and injuries can lead to corneal blindness, one of the major causes of blindness. Alternative treatments are developed because of the limitation of traditional corneal transplantation. These treatments benefit from the study of the molecular basis of corneal development and regeneration. However, corneal development is not fully understood. The purpose of this work was to elucidate the gene expression profiles during pNC migration and to examine the function of a highly regulated gene, Nephronectin (NPNT), during corneal formation. By performing RNA-seq analysis comparing pNC to its derived corneal structures, I analyzed differentially expressed genes and examined differentiated pathways during corneal formation. This project summarized the transcriptional regulation that happens at three levels: signaling pathways, transcription factors, and the downstream endothelial and stromal genes, providing gene candidates involved in corneal formation for future studies. From the RNA-seq analysis, I identified novel upregulation of NPNT among the extracellular matrix (ECM) proteins of the cornea. NPNT has been studied in other developmental processes but has not been linked to the corneal formation. Here, I found that NPNT is distributed in the primary stroma during pNC migration. Its receptor, Integrin (ITG) α8, is expressed in the pNC that migrate on the primary stroma. Thus, I hypothesized that NPNT interacts with ITGα8β1 to promote pNC migration during corneal development. I performed functional studies by the RCAS-RNAi system. The knockdown of either NPNT or ITGα8 resulted in the reduction of corneal stromal thickness. Further studies revealed that NPNT overexpression upregulated cell numbers in the corneal stroma but did not increase cell proliferation. Inhibition of ITGα8 in vivo and in vitro both reduced pNC migration. Together, the functional studies link NPNT/ITGα8β1 signaling to pNC migration during corneal formation. This study reveals a previously unknown ECM-receptor pathway in corneal formation, suggesting a potential gene target or culture matrix in corneal development and regeneration. Collectively, this work depicts the transcription profiles during chick embryonic corneal development and investigates the function of a candidate ECM protein, NPNT, in this process. This comprehensive analysis served as a foundation of the molecular mechanisms underlying pNC migration, proliferation, and differentiation, providing potential clinical targets during corneal development and induction signals for corneal regeneration.