This review article focuses on the targeting of cancer stem cells (CSCs) for cancer therapy. CSCs, first identified in leukemia in 1994, are characterized by their self-renewal capacity and differentiation potential, contributing to tumor recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. The biological activities of CSCs are regulated by several pluripotent transcription factors, such as OCT4, Sox2, Nanog, KLF4, and MYC, as well as various intracellular signaling pathways, including Wnt, NF-κB, Notch, Hedgehog, JAK-STAT, PI3K/AKT/mTOR, TGF/SMAD, and PPAR, and extracellular factors like vascular niches, hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, and exosomes. Molecules, vaccines, antibodies, and CAR-T cells have been developed to specifically target CSCs, with some already in clinical trials. The article summarizes the characterization and identification of CSCs, the major factors and pathways regulating CSC development, and potential targeted therapies for CSCs.This review article focuses on the targeting of cancer stem cells (CSCs) for cancer therapy. CSCs, first identified in leukemia in 1994, are characterized by their self-renewal capacity and differentiation potential, contributing to tumor recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. The biological activities of CSCs are regulated by several pluripotent transcription factors, such as OCT4, Sox2, Nanog, KLF4, and MYC, as well as various intracellular signaling pathways, including Wnt, NF-κB, Notch, Hedgehog, JAK-STAT, PI3K/AKT/mTOR, TGF/SMAD, and PPAR, and extracellular factors like vascular niches, hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, and exosomes. Molecules, vaccines, antibodies, and CAR-T cells have been developed to specifically target CSCs, with some already in clinical trials. The article summarizes the characterization and identification of CSCs, the major factors and pathways regulating CSC development, and potential targeted therapies for CSCs.