Cancer stem cells are a subpopulation of cancer cells characterized by self-renewal and differentiation potential, as well as high tumorigenicity. They play a major role in cancer initiation, progression, and metastasis. Therefore, they are a major focus in the development of next-generation cancer therapies. This review aims to give a comprehensive overview of the biology of cancer stem cells, including their heterogeneity and metabolic plasticity, as well as their dynamic interaction with the tumor microenvironment, including hypoxia, immune modulation, and extracellular matrix signaling. In addition, it aims to provide an overview of some of the major pathways involved in cancer stem cell regulation, including the Wnt/β-catenin, Notch, Hedgehog, and PI3K/Akt/mTOR pathways. This review also aims to give an overview of the recent advances in cancer therapy, including the development of small molecules, epigenetic drugs, natural compounds, biologics, and immunotherapies targeting cancer stem cells. Despite the advances in cancer therapy, cancer stem cell heterogeneity and therapy resistance have proved to be major challenges in cancer treatment. Therefore, it is crucial to understand the biology of cancer stem cells and their interactions with the tumor microenvironment to develop effective, durable, and targeted cancer therapies.