Spermatogonial stem cells (SSCs) divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells (hdFs). Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen (SSEA)-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the 'niche' that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.