Stromal cells are thought to generate specific regulatory microenviroments or “niches” that control stem cell behavior. Characterizing stem cell niches in vivo remains an important goal that has been difficult to achieve. The individual ovarioles of the Drosophila ovary each contain about two germ line stem cells that maintain oocyte production. Here we show that anterior ovariolar somatic cells comprising three cell types act as a germ line stem cell niche. Germ line stem cells lost by normal or induced differentiation are efficiently replaced, and the ability to repopulate the niche increases the functional lifetime of ovarioles in vivo. Our studies implicate one of the somatic cell types, the cap cells, as a key niche component.