DIP2 protein contains three members: DIP2A, DIP2B and DIP2C, and are broadly expressed in the nervous system from Drosophila to human during embryonic development. Dip2c gene-associated mutations have been reported in tumors and neuronal diseases. However, the role of Dip2c in the context of mouse embryonic stem (mES) cells has not been explored. To investigate the biological function of Dip2c during early embryo development, we generated Dip2c-/- mES line using a CRISPR/Cas9 system. This cell line has contributed to further investigation of molecular mechanism of Dip2c during cell differentiation, as well as a cell model for screening for neurogenic drug and cancer clinical cure.