The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three-colour immunofluorescence. In normal donor bone marrow the assay enables simultaneous identification of immature (CD13+, CD14-, CD66-), intermediate (CD13+, myelopoietic differentiation stages through the exclusion of CD14+ monocytic cells. In the diagnosis and longitudinal follow-up of AML patients the assay was of value in the fast determination of remission state. In MDS, the immature myeloid component could be distinguished in patients defined according to the FAB classification with the possibility of identifying aberrant phenotypes, the assay should also be of interest in other myeloproliferative disorders. Moreover, because it is easy to perform, time-saving, and yields comparable results to single antibody reactivity controls, it can replace more tedious and less-informative flow cytometric immunophenotyping procedures.