Stem cells have the ability to self-renew or differentiate into specific lineages. Discovering the means by which they accomplish these feats brings us closer to realizing the promise of these cells in regenerative medicine. In this study we analyzed the expression of a class of small RNA molecules, microRNAs (miRNAs), in multiple adult tissue stem cells before and after their exit from the stem cell state. MiRNAs repress multiple genes and provide an additional layer of gene regulation prior to protein translation, and we have yet to study those miRNAs utilized by stem cells. Our analyses revealed miRNAs common or unique to blood, muscle, and neural stem cell populations that change during the early differentiation process. We identified a subset of miRNAs that increase during the exit from a self-renewing state in multiple adult tissue stem cells and were also changed by mutations and cancers that altered stem cell properties. When we forced the expression of these miRNAs in stem cells, they diminished their stem cell activity and targeted genes required for stem cell self-renewal. This study lays a foundation for understanding the role of miRNAs in stem cells and demonstrates that adult tissue stem cells up-regulate miRNAs that target key self-renewal genes as they differentiate.