Spermatogenesis originates from spermatogonial stem cells (SSCs). Development of the spermatogonial transplantation technique in 1994 provided the first functional assay to characterize SSCs. In 2000, glial cell line-derived neurotrophic factor was identified as a SSC self-renewal factor. This discovery not only provided a clue to understand SSC self-renewing mechanisms but also made it possible to derive germline stem (GS) cell cultures in 2003. In vitro culture of GS cells demonstrated their potential pluripotency and their utility in germline modification. However, in vivo SSC analyses have challenged the traditional concept of SSC self-renewal and have revealed their relationship with the microenvironment. An improved understanding of SSC self-renewal through functional assays promises to uncover fundamental principles of stem cell biology and will enable us to use these cells for applications in animal transgenesis and medicine. © 2013 by Annual Reviews. All rights reserved.