Cancer stem cells (CSCs) are a subpopulation of cancer cells that have the ability to self-renew and to generate differentiated cells of various lineages. Due to their specific morphological and biological features, they are often resistant to therapy and in turn lead to metastasis and cancer recurrence. Because of their crucial roles in carcinogenesis and patient prognosis, identification and isolation of CSCs have become an important part of improved cancer management regime. Isolation, characterization, and development of targeted therapy against CSCs have potential efficacy in treating esophageal cancer. In addition, CSCs can act as a predictive tool for chemoradiotherapy response in esophageal adenocarcinoma. Different methods including functional assays, cell sorting using various intracellular, and cell surface markers and xenotransplantation techniques are used for the identification and separation of CSCs in different cancers. None of these methods solely can guarantee complete isolation of CSC population, thus a combination of methods could be used for reliable detection and isolation of CSCs. Here, we describe the identification and isolation of CSCs from esophageal adenocarcinoma cells by cell sorting after Hoechst 33342 staining followed by in vitro functional assays, and in vivo xenograft techniques.