Practical procedures for sorting and analysis of leukemia stem cells (LSCs) are to improve our understanding of chronic myelogenous leukemia (CML). Here, we present a detailed magnetic-bead-based sorting and flow-cytometry-based analysis protocol for LSCs in BCR-ABL-driven CML mice. We describe steps for sorting and functional analysis of BCR-ABL-expressing c-Kit+ cells (GFP+c-Kit+) from CML mice as well as antibody staining and gating strategies for characterization of leukemia stem/progenitor cells and myeloid leukemia cells. For complete details on the use and execution of this protocol, please refer to Liu et al. (2022).1.Keywords: Cell isolation; Flow Cytometry/Mass Cytometry; Model Organisms; Stem Cells.