Generation of functional eggs from pluripotent stem cells in culture would have impacts on reproductive biology and regenerative medicine. We have recently established a novel culture system in which mouse pluripotent stem cells differentiate to mature oocytes in a manner similar to oogenesis in vivo. Importantly, the in vitro-generated oocytes are capable of full-term development. This protocol consists of a culture method that can be mainly divided into four periods: primordial germ cell-like cell (PGCLC) derivation, primary oocyte differentiation (in vitro differentiation: IVDi), oocyte growth to germinal vesicle (GV) oocytes (in vitro growth: IVG) and oocyte maturation (in vitro maturation: IVM). In each process, a robust number of mouse oocytes can be generated in a dish.