Tumor-initiating cells (T-ICs) are a subpopulation of chemoresistant tumor cells that have been shown to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of new therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. With this model, we found CD24 to be upregulated in residual chemoresistant tumors when compared with bulk tumor upon cisplatin treatment. CD24⁺ HCC cells were found to be critical for the maintenance, self-renewal, differentiation, and metastasis of tumors and to significantly impact patients' clinical outcome. With a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through STAT3-mediated NANOG regulation. Our findings point to a CD24 cascade in liver T-ICs that may provide an attractive therapeutic target for HCC patients.