The cancer stem cell hypothesis is an appealing concept to account for intratumoral heterogeneity and the observation that systemic metastasis and treatment failure are often associated with the survival of a small number of cancer cells. Whilst in vivo evidence forms the foundation of this concept, in vitro methods and reagents are attractive as they offer opportunities to perform experiments that are not possible in an animal model. While there is abundant evidence that existing cancer cell lines are not reliable models of tumor heterogeneity, recent advances based on well validated novel cancer cell lines established de novo in defined serum-free media are encouraging, particularly in the study of glioblastoma multiforme. In this chapter we wish to broadly outline the process of establishing, characterizing, and managing novel cancer cell lines in defined serum-free media, and discuss the limitations and potential opportunities that may arise from these model systems.