The fallopian tube (FT) is an important reproductive organ in females. Ample evidence suggests that the distal end of FT is the original site of high-grade serous ovarian carcinoma (HGSC). FT may suffer from repeated injury and repair stimulated by follicular fluid (FF); however, this hypothesis has not been examined. In fact, the molecular mechanism of homeostasis, differentiation, and the transformation of fallopian tube epithelial cells (FTECs) resulting from the stimulation of FF are still enigmatic. In this study, we examined the effects of FF along with factors present in the FF on a variety of FTEC models, including primary cell culture, ALI (air–liquid interface) culture, and 3D organ spheroid culture. We found that FF plays a similar role to estrogen in promoting cell differentiation and organoid formation. Moreover, FF significantly promotes cell proliferation and induces cell injury and apoptosis in high concentrations. These observations may help us to investigate the mechanisms of the initiation of HGSC.