Objective: The fate of spermatogonial stem cells, as the foundation of spermatogenesis are tightly regulated by some intrinsic factor and extrinsic factors from their nich. Along the biological understanding of these mechanisms, a major concern is improving the efficiency, evaluation of the safety of their clinical application. Because the mechanisms that involved in human spermatogenesis are complex and unknown, this study was designed to providing an appropriate in vitro system to proliferate and enrichment of hSSC. Materials and Methods: The isolation of spermatogonial stem cells (SSCs) were performed using two step enzymatic digestions and plating method. The identity of the SSCs and sertoli cells was confirmed through immunocytochemistry. We were designed 4 various culture system: co-culture with patient own Sertoli cells, co-culture with normal sertoli cells obtained from a person with normal spermatogenesis and culture of SSC on un-coated dishes and culture of testis cells suspension. The number and diameter of colonies were evaluated during the 3 weeks of culture. The identity of the SSC and sertoli cells was confirmed by immunocytochemistry against PLZF, GFR-a1and vimentin. The expression profile of the several germ stem cell specific and pluripotency markers were assessed using quantitative RT-PCR. Results: Significant differences were observed between the four groups (p<0.05), with higher mean in number and diameter of colonies for co-culture with testis cells suspension in compare with other groups (p<0.05). Analysis of marker expression revealed that there are higher expression of germ stem cell markers and lower expression of pluripotency markers in co-culture with Sertoli cells. Conclusion: Our findings demonstrated that adult coculturing of SSCs with Sertoli cells can influence spermatogonial proliferation in vitro.