Different cancer stem cell (CSC) populations can be found in many types of cancer, including squamous cell carcinoma (SSC). Diverse reports showed that CSC play a crucial role in the relapse of different types of cancer. CSC sustains tumor growth due to their capacity to self-renew and their potential to initiate secondary tumors with metastatic cancer features. Therefore, the development of methods for the isolation of CSC is a key step to explore the mechanisms underlying CSC maintenance. In this chapter, we provide a method for isolating CSC from cutaneous SSC using immunofluorescence labeling to allow the specific purification of CSC by fluorescence-activated cell sorting (FACS). This method is based on the use of CSC membrane markers, allowing as well the isolation CSC from different mouse strains.