While the mesonephros persists in the adult mouse ovary and contributes to folliculogenesis, its inductive role in oogonial stem cell (OSC) differentiation remains uncertain. In this study, we investigated whether mesonephros-derived cells direct OSCs toward follicle-like structures in vitro. OSCs were co-cultured with mesonephros cells, and morphological assessments and factor expression analyses were conducted at multiple time points. On day 14 of co-culture, 10× Genomics single-cell and bulk RNA sequencing were performed to profile transcriptomic changes, while renal capsule transplantation was conducted to evaluate the functional potential of co-cultured clusters in recipient mice. Transcriptomic analyses revealed a robust expression of mesonephros-specific genes and a significant enrichment of PI3K/AKT pathway-associated genes in co-cultured cells. Notably, transplanted clusters exhibited signs of estrogen secretion with maintained graft integrity. Collectively, these findings demonstrate that the mesonephros promotes OSC differentiation into follicle-like structures, with the PI3K/AKT pathway identified as a candidate mediator of this process. Our study provides novel insights into the developmental regulation of ovarian folliculogenesis and offers a valuable experimental model for future investigations.