The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture system, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of stem cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ cell differentiation gene expression.