Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation1,2. The whole process, therefore, has never been reproduced in vitro in mammals3,4,5, nor in any other species with a very few exceptions in some particular types of fish6,7. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media.