Background: A “indirect co-culture using mesh” system is commonly employed to maintain spermatogenesis in cancer patients undergoing chemotherapy and radiation. This study aimed to investigate the co-culturing of mouse spermatogonial stem cells (SSCs) with human amniotic mesenchymal stem/stromal cells (hAMSCs) in an optimized environment. Methods: SSCs from 3-6-day-old mice (n=10) were indirectly co-cultured with hAMSCs via mesh for two weeks. Three groups evaluated: control, SSCs with conditioned media, and SSCs indirectly co-cultured with hAMSCs. Gene expression analyzed for Plzf, c-kit, Sycp3, and Prm1. Immunohistochemistry assessed Plzf, and flow cytometry evaluated c-kit and Plzf. Results: Showed a twofold increase in Plzf-positive cells after 14 days of culture (76.47%, P≤0.05), with a significant elevation in Plzf gene expression observed in the conditioned media group (188.1 ± 65%, P≤0.05). Conversely, the expression of the c-kit gene decreased significantly in both the conditioned media and “indirect co-culture using mesh” groups. Notably, Sycp3 and Prm1 expression levels significantly increased in the conditioned media group compared to the control. These findings suggest the potential of conditioned media as a novel feeder for promoting in vitro mouse spermatogenesis. Conclusion: Our results demonstrate that the inclusion of growth factors, such as GDNF and BMP-4, along with conditioned media and an “indirect co-culture using mesh” system utilizing meshes with SSCs, significantly enhances SSC proliferation and differentiation. The optimized conditions media provided by hAMSCs offer a superior feeder compared to traditional “indirect co-culture using mesh” systems for promoting both the proliferation and differentiation of SSCs.