AbstractAcute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all‐trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per‐cell quantitative image analysis for statistically large sample sizes, enabling precise and operator‐independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment. © 2012 International Society for Advancement of Cytometry