The cornea and sclera are distinct adjacent tissues, yet their stromal cells originate from common neural crest cells (NCCs). Sclerocornea is a disease characterized by an indistinguishable boundary between the cornea and sclera. Previously, we identified a RAD21 mutation in a sclerocornea pedigree. Here, we investigated the impacts of RAD21 on NCC activities during eye development. RAD21 deficiency caused upregulation of PCDHGC3. Both RAD21 knockdown and PCDHGC3 upregulation disrupted the migration of NCCs. Transcriptome analysis indicated that WNT9B had 190.9-fold higher expression in scleral stroma than in corneal stroma. WNT9B was also significantly upregulated by both RAD21 knockdown and PCDHGC3 overexpression, and knock down of WNT9B rescued the differentiation and migration of NCCs with RAD21 deficiency. Consistently, overexpressing wnt9b in Xenopus tropicalis led to ocular developmental abnormalities. In summary, WNT9B is a determinant factor during NCC differentiation into corneal keratocytes or scleral stromal cells and is affected by RAD21 expression. Display omitted •Established a stable differentiation protocol from hESCs to corneal keratocytes•RAD21 deficiency affected the proliferation and migration ability of NCCs•Increased scleral markers after RAD21 knockdown during NCC differentiation to cornea•WNT9B is a crucial mediator during ocular NCC differentiation Cell biology; Developmental biology