BMI1-expressing cancer stem cells (CSCs) play a key role in the development, progression, therapy resistance, recurrence, and metastasis of head and neck squamous cell carcinoma (HNSCC). Here, we present a chemically-induced HNSCC mouse model, genetically and pathologically similar to human HNSCC. This protocol describes how to use genetic lineage tracing based on the Cre-loxP recombination strategy, which allows us to study the regulation and targeting of BMI1+ CSCs in primary tumors and lymph node metastases. For complete details on the use and execution of this protocol, please refer to Chen et al. (2017) and Jia et al. (2020).