Spermatogonial stem cells (SSCs) in the testes are a new target for germline modification. With the development of an in vitro culture system and spermatogonial transplantation technique, SSCs can now be manipulated and used as an alternative to embryonic stem cells for knockout mice production. The genetic and epigenetic stability of SSCs provide new possibilities for the application of germline mutagenesis in a wide range of animals.