Abstract Genome maintenance in the germline is vital for fertility and the health of offspring. Although DNA damage response (DDR) and repair has been well characterized in meiocytes, the DDR pathway(s) that function in primordial germ cells (PGCs), which are the precursors to sperm and eggs, have not been identified. Fanconi anemia (FA) is a genomic instability syndrome associated with PGC depletion and infertility in addition to some other devastating clinical manifestations such as bone marrow failure and cancer predisposition. The FA pathway is a major DDR pathway known to function in double-strand DNA break repair. The signaling mechanisms underlying PGC depletion and infertility in FA patients or mouse models are not known. We recently developed Flag- and hemagglutinin-tagged Fancd2 knock-in mice that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DDR partners, we observed several proteins of the germ-cell-specific Prmt5/piRNA pathways orchestrating the repression of transposable elements (TEs). Deletion of Fancd2 resulted in decreased protein levels of the Prmt5/piRNA factors, massive upregulation of TEs, PGC depletion, and led to defective spermatogenesis and oogenesis in Fancd2-null (Fancd2-KO) mice. These preliminary studies suggest that in addition to its well-established DNA repair roles, Fancd2 and the FA pathway has an in vivo TE silencing function in early-stage germ cells. We hypothesize that the FA pathway is essential for germline integrity involving a pathway hierarchy in which the FA core signals to Fancd2, which then guides Prmt5 and piRNA in TE silencing. The goals of the project are to establish functional interaction between the FA pathway and germ cell- specific TE silencing machinery in safeguarding the germline genome, and to define Fancd2 as a crucial regulator of this vital epigenetic programming during germ cell development. The project presents an innovative study aimed at linking a major DDR pathway to germline genome maintenance in early-stage germ cells. The knowledge gained from the proposed study will not only improve mechanistic understanding of the molecular collaboration between the FA DDR pathway and the Prmt5/piRNA pathway in the context of TE repression, but also lead to a new avenue of research designed to target these interacting pathways for developing innovative therapeutic strategies for reproductive diseases such as infertility and birth defects.