Flow cytometry (FCM) is an established ancillary technique applied to the diagnosis of hematological malignancies and for measurement of DNA content. In recent years, the number of fluorochrome-conjugated antibodies available for immnuofluorescence and FCM has greatly increased, making it possible to evaluate this technique in other diagnostic contexts, as well as to study a range of biological processes. Serous effusions are optimal for studies using FCM as they consist of viable cells in suspension. Molecules that have been studied for their expression and clinical role in effusions in recent years participate in central cellular functions, including adhesion, proliferation, cellular metabolism, and apoptosis, as well as intracellular signaling. FCM can further be applied to quantitative analysis of molecules related to chemotherapy response, which, together with apoptosis, may represent an important tool for evaluating treatment response and prognosis in advanced and/or recurrent cancer. As targeted therapy assumes an ever-growing role in the treatment of metastatic cancer, the ability to study living cells in effusions or fine-needle aspirates is becoming more important. This article reviews the currently available data in this area of cytopathology. Diagn. Cytopathol. 2012;40:525–535. © 2012 Wiley Periodicals, Inc.