Objective: Spermatogonial stem cells (SSCs) are unique population of adult stem cells in mammalian testes which continuously provide gametes and transfer genetic material to the next generation. Various feeder layers have been tested to support the in vitro culture of SSCs; however age effect of feeder cells has been remained a controversial issue. This study was initiated to compare Sertoli cells derived from neonatal and adult mice to examine age effect of Sertoli cells in the maintenance and proliferation of mouse SSCs in vitro. Materials and Methods: SSCs were isolated from testes of 6 day-old mice and culture in vitro at the presence of Glial-derived neurotrophic factor (GDNF) for 10 days and then transferred to Sertoli cells isolated by DSA lectin from neonatal (6 day-old) and adult (6-8 week-old) mice. After 5 days area and number of SSC colonies were measured. Immunostaining was used to detect expression of spermatogonial markers including α6/ß1-Integrin C-kit and Oct-4. In addition SSC colonies were harvested at day 5 and the percentage of α6/ß1-Integrin-positive cells was measured by flowcytometery. Transplantation assay was used to confirm the stemness of spermatogonial cells. Results: Immunostaining analysis showed that our culture system contained SSC colonies as they were positive for α6-Integrin ß1-Integrin and Oct-4 and negative for C-kit. In addition these stem cells were functional as they were able to migrate to seminiferous basal membrane after transplantation to testes of busulfan-induced infertile adult miceResults showed 5 days after co-culture of SSCs with Sertoli cells the area and number of colonies and number of cells in each colon were significantly higher on Immature Sertoli cells. Moreover results showed that colony efficiency of cultured SSCs on Immature Sertoli cells was significantly higher than other two groups. Flowcytometry analysis revealed that there was significant increase in the number of α6-Integrin-positive cells and ß1-Integrin-positive cells in the culture with Immature Sertoli cells (72.9 ± 14.9 %)(67.4 ± 7.7) in contrast to culture with Mature Sertoli cells (29.5 ± 6.5 %)(52.9± 1.9). Conclusion: In conclusion the number of SSCs was higher in co-culture with Immature Sertoli cells. It could be referred to difference in microenvironments that Immature and Mature Sertoli cells provide for in vitro culture of SSCs. As SSCs was derived from neonatal mice another interpretation is that more suitable culture condition could be obtained when SSCs and feeder Sertoli cells are derived from mice in the same age; either from neonatal mice or from adult ones. Further studies would confirm these hypotheses.