Cancer stem cells (CSCs) are critical targets for cancer therapy due to their ability to self-renew, differentiate, and contribute to tumor recurrence, metastasis, and resistance to treatment. Identified in leukemia in 1994, CSCs are characterized by their self-renewal capacity, differentiation potential, and expression of markers like CD34+ and CD38-. They are regulated by transcription factors (e.g., OCT4, Sox2, Nanog, KLF4, MYC) and signaling pathways (e.g., Wnt, NF-κB, Notch, Hedgehog, JAK-STAT, PI3K/AKT/mTOR, TGF/SMAD, PPAR) as well as extracellular factors like vascular niches, hypoxia, and exosomes. Various molecules, vaccines, antibodies, and CAR-T cells have been developed to target CSCs, with some undergoing clinical trials. This review summarizes the characterization, identification, and regulation of CSCs, and discusses potential targeted therapies. CSCs are heterogeneous and play a crucial role in tumor initiation, metastasis, and resistance to treatment. They can be isolated using techniques like FACS, MACS, and colony-forming assays. Key transcription factors like Oct4, Sox2, Nanog, KLF4, and MYC regulate CSC growth and survival. Major signaling pathways such as Wnt, Notch, and Hedgehog are involved in CSC self-renewal, differentiation, and metastasis. Aberrant activation of these pathways is associated with cancer progression. Understanding these mechanisms is essential for developing effective therapies targeting CSCs.Cancer stem cells (CSCs) are critical targets for cancer therapy due to their ability to self-renew, differentiate, and contribute to tumor recurrence, metastasis, and resistance to treatment. Identified in leukemia in 1994, CSCs are characterized by their self-renewal capacity, differentiation potential, and expression of markers like CD34+ and CD38-. They are regulated by transcription factors (e.g., OCT4, Sox2, Nanog, KLF4, MYC) and signaling pathways (e.g., Wnt, NF-κB, Notch, Hedgehog, JAK-STAT, PI3K/AKT/mTOR, TGF/SMAD, PPAR) as well as extracellular factors like vascular niches, hypoxia, and exosomes. Various molecules, vaccines, antibodies, and CAR-T cells have been developed to target CSCs, with some undergoing clinical trials. This review summarizes the characterization, identification, and regulation of CSCs, and discusses potential targeted therapies. CSCs are heterogeneous and play a crucial role in tumor initiation, metastasis, and resistance to treatment. They can be isolated using techniques like FACS, MACS, and colony-forming assays. Key transcription factors like Oct4, Sox2, Nanog, KLF4, and MYC regulate CSC growth and survival. Major signaling pathways such as Wnt, Notch, and Hedgehog are involved in CSC self-renewal, differentiation, and metastasis. Aberrant activation of these pathways is associated with cancer progression. Understanding these mechanisms is essential for developing effective therapies targeting CSCs.