Shortly after the discovery of cancer stem cells in acute myeloid leukemia, cancer stem cells in several cancer types were discovered, including brain cancer. Brain tumor stem cells (BTSCs), initially characterized by the expression of CD133, are a population of cells that can proliferate and form spheres in vitro and form tumors in vivo. Due to the belief that they are treatment-resistant and seed-recurrent diseases, they are an ideal preclinical model that can be used to study brain cancer. In this chapter, we describe how to process, maintain, and propagate a BTSC culture from brain tumor tissue samples.