Flow cytometry (FCM) is a powerful technology that can provide rapid multiparameter analysis of single cells. With the significant improvement in instrumentation and reagents during the past several decades, multicolor FCM can quickly analyze lymphoid and hematopoietic cells and plays a critically important role in the diagnosis of most hematolymphoid neoplasms. Being able to rapidly identify abnormal blast populations and give blast lineage classification, FCM study is indispensable for the diagnosis and posttreatment monitoring of acute leukemia. With the ability of highly efficient immunotyping and clonality assessment for mature B-cells and mature αβ T-cells, FCM can quickly characterize the phenotype of an abnormal lymphoid population, make the diagnosis, or assist in the classification of a mature lymphoid neoplasm. In addition, FCM study can sensitively detect the phenotypic abnormalities of myeloid cells and erythroid precursors in patients with myelodysplastic syndrome or other myeloid neoplasms. FCM can readily measure DNA content and determine the DNA ploidy of a cell population, which is useful for making a diagnosis and/or predicting prognosis. FCM study is also useful in the identification of the malignant cells of non-hematologic malignancies, especially in liquid specimens such as bone marrow, body fluid, and fine needle aspiration. Correlation with histomorphologic features, clinical information, and sometimes cytogenetic/molecular findings is always required for accurate interpretation of FCM results. This chapter provides an overview of the principles and some advances of FCM in the diagnosis of hematological neoplasms and other cancers.