: Spermatogenesis depends on a population of cells called spermatogonial stem cells, which self‐renew to support male reproduction throughout life. In 2003, the long‐term culture of spermatogonial stem cells of mice proved to be successful. In the presence of glial cell‐line‐derived neurotrophic factor, germline stem (GS) cells were established from postnatal mouse testis. These cells proliferated over a 2‐year period (>10⁸⁵‐fold) and restored fertility to congenitally infertile recipient mice following transplantation into the seminiferous tubules. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, the GS cells retained their euploid karyotype and androgenetic imprint during the 2‐year experimental period, and they produced normal fertile offspring. Mutagenization of the GS cells was successful using gene trapping and gene targeting vectors to produce homozygous knockout offspring, thereby providing a new approach to germline modification. In the course of the gene targeting experiments, establishment of embryonic‐stem (ES)‐like cells was also successful [i.e., multipotent germline stem (mGS) cells] from postnatal mouse testis. These mGS cells were phenotypically similar to the ES/embryonic germ cells, except for their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under the conditions used to induce the differentiation of the ES cells, and the mGS cells formed germline chimeras when injected into blastocysts. These new spermatogonial stem‐cell lines will be useful for studying the mechanism of spermatogenesis, and they have important implications for developing new transgenic or medical technologies.