Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.