Follicular development commences with the formation of primordial follicles, which begins with the differentiation of pluripotent ovarian somatic cells into early granulosa cells and their apposition to the oocytes in the egg nest. The process of primordial follicle morphogenesis and factors affecting the formation and development of primordial follicles can be examined in vitro using fetal ovaries in organ culture. The functions of candidate genes involved in primordial folliculogenesis can be examined using siRNA or shRNA, which can knockdown specific mRNA targets at specific time points. Here, we describe the organ culture protocol for fetal hamster ovary with GPR30 siRNA as an example. The method to morphologically analyze follicular development is also discussed.