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Hematopoiesis is maintained throughout life from the hematopoietic stem cell niche in which hematopoietic stem cells and lineage-specific hematopoietic progenitors (HSPCs) reside and regulate hematopoiesis. Meanwhile, HSPCs behavior is modulated by both cell intrinsic (e.g., transcriptional factors) and cell extrinsic (e.g., cytokines) factors. Dysregulation of these factors can alter HSPCs function, leading to disrupted hematopoiesis, cellular changes, and subsequent hematological diseases and malignancies. Moreover, it has been reported that chromosomal aberration (CA) in HSPCs following exposure to carcinogenic or genotoxic agents can initiate leukemia stem cells (LSCs) formation which lays a fundamental mechanism in leukemogenesis. Despite reported studies concerning the chromosomal integrity in HSPCs, CA analysis in lineage-specific HSPCs remains scarce. This indicates a need for a laboratory technique that allows the study of CA in specific HSPCs subpopulations comprising differential hematopoietic lineages. Thus, this chapter focuses on the structural (clastogenicity) and numerical (aneugenicity) form of CA analysis in lineage-specific HSPCs comprised of myeloid, erythroid and lymphoid lineages.In this protocol, we describe how to perform CA analysis in lineage-specific HSPCs derived from freshly isolated mouse bone marrow cells (MBMCs) using the combined techniques of colony-forming unit (CFU) and karyotyping. Prior to CA analysis, lineage-specific HSPCs for myeloid, erythroid, and lymphoid were enriched through colony-forming unit (CFU) assay. CFU assay assesses the proliferative ability and differentiation potential of an individual HSPC within a sample. About 6 to 14 days of cultures are required depending on the type of HSPCs lineage. The optimal duration is crucial to achieve sufficient colony growth that is needed for accurate CFU analysis via morphological identification and colony counting. Then, the CA focusing on clastogenicity and aneugenicity anomalies in respective HSPCs lineage for myeloid, erythroid and Pre-B lymphoid were investigated. The resulted karyotypes were classified according to the types of CA known as Robertsonian (Rb) translocation, hyperploidy or complex. We believe our protocol offers a significant contribution to be utilized as a reference method for chromosomal analysis in lineage-specific HSPCs subpopulations., (© 2023. Springer Science+Business Media, LLC.)