Abstract: Zebrafish has emerged as a valuable model for investigating germ cell biology and reproduction. The AB strain of zebrafish is proposed to have a polygenic sex determination system and the majority of males develop juvenile ovaries before committing to male fate. In the species with chromosomal sex determination system, it is widely accepted that the gonadal somatic cells play a pivotal role in determining the fate of germ cells. Interestingly, it has been found that the loss of germ cells leads to masculinization in zebrafish, implying that germ cells harbor an intrinsic feminization signal. However, which signal initiates oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene which acts as an indispensable signal for germ cells to enter oogenesis. We first found that the foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further study revealed that the loss of foxl2l hindered the initiation of oocyte-specific meiosis and obstructed the entry into oogenesis, resulting in premature spermatogenesis during early gonadal development. Furthermore, we showed that while the mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants resulted in a complete blockage of oogenesis, with a large proportion of germ cells arrested in the state of germline stem cells, highlighting the essential role of foxl2l in oogenesis. Overall, our study revealed the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.