Spermatogonial stem cells (SSCs) are the most primitive spermatogonia in the testis and have an essential role to maintain highly productive spermatogenesis by self-renewal and continuous generation of daughter spermatogonia that differentiate into spermatozoa, transmitting genetic information to the next generation. Since the 1950s, many experimental methods, including histology, immunostaining, whole-mount analyses, and pulse-chase labeling, had been used in attempts to identify SSCs, but without success. In 1994, a spermatogonial transplantation method was reported that established a quantitative functional assay to identify SSCs by evaluating their ability to both self-renew and differentiate to spermatozoa. The system was originally developed using mice and subsequently extended to nonrodents, including domestic animals and humans. Availability of the functional assay for SSCs has made it possible to develop culture systems for their ex vivo expansion, which dramatically advanced germ cell biology and allowed medical and agricultural applications. In coming years, SSCs will be increasingly used to understand their regulation, as well as in germline modification, including gene correction, enhancement of male fertility, and conversion of somatic cells to biologically competent male germline cells.