Background: According to the 2008 WHO classification, the use of flow cytometry (FC) has an important role in the diagnosis of acute leukemia as it objectively decides on the leukemic nature and lineage of the blast cell population whether it is of myeloid, lymphoid or ambiguous origin. Aims of the study: Using multicolor FC to evaluate the expression of CD7, CD11c, CD13, CD14, CD19, CD33, CD34, CD45, CD64, CD117 and MPO in adult patients with morphologically diagnosed AML; CD1a, CD2, CD3, CD5, CD7, CD10, CD13, CD19, CD20, CD22, CD33, CD34, CD45, CD79a, cμ and TdT in patients with morphologically diagnosed ALL. To evaluate the threshold for MPO expression within blast/immature cells by FC and IHC for defining myeloid lineage to the blasts/leukemia. To suggest an immunophenotypic paradigm for the diagnosis and classification of acute leukemia in adults using multicolor FC. Patients, materials and methods: This cohort study included 86 adult patients, 49 males and 37 females, with newly diagnosed acute leukemia, 61 patients with AML, 24 patients with ALL and 1 patient with acute undifferentiated leukemia, from April 2012 to May 2013. For each patient, peripheral blood and bone marrow aspirate (BMA) samples were requested/withdrawn to perform full blood count, peripheral blood film and BMA morphology and multicolor FC analysis, for the expression of designated markers in patients with newly, morphologically, diagnosed acute leukemia, was carried out using 2-laser, 4-color, 6-parameter PARTEC Cube6 with De Novo FCS Express 4 software on 31 BMA samples collected from private laboratory-Baghdad; and using 3-laser, 8-color, 10- parameter BD FACSCANTO II with De Novo FACS DIVA 6.1.3 on 55 BMA samples from the Hammersmith Hospital-London. For a subpopulation of patients, bone marrow trephine biopsy (BMTB) with immunohistochemistry staining for MPO was performed. Results: Using multicolor the blast gate of CD45 versus SSC plot in all patients with T and B ALL and in ˃98% patients with AML with a statistically highly significant difference. CD7 and CD19 were aberrantly expressed in 27.9% and 11.5% of patients with AML, and were universally expressed in patients with T and B ALL respectively with highly significant difference in percentage of expression that is in favor of ALL. CD13 and CD33 were expressed in 93.4% and 85.2% of patients with AML, and were aberrantly expressed in 20.8% and 12.5% of patients with ALL with highly significant difference that is in favor of AML. MPO, CD34 and CD117 were expressed in 95.4%, 96.7% and 95% of patients with AML. Co-expression of 2 or more monocytic markers was present in 20% of patients with AML. Range of blast cells in T ALL, assessed by morphology, was 42-97%; TdT and CD3 were universally expressed and CD34 was expressed in 90% of patients with T ALL. Range of blast cells in B ALL, assessed by morphology, was 68-99%; TdT and CD19 were universally expressed in patients with B ALL. The comparison of MPO expression in myeloblasts assessed by FC on BMA with expression assessed by IHC on BMTB shows a direct significant relation between them (p-value 0.0246). Conclusions: For screening and diagnosis of acute leukemia, start with serial gating using FSC versus SSC, then CD45 and CD34 versus SSC, then using surface markers that are highly sensitive for each subtype of acute leukemia, CD13 for AML, CD7 for T ALL and CD19 for B ALL. For confirming the nature of cells as being myeloblasts, test for cMPO and CD33 and/or CD117 expression. For identification of patients with AML with monocytic differentiation, after proper serial gating to isolate the possible leukemic myeloblasts/monoblasts/promonocytes population, further gating using CD14 then testing the resulting population for CD11c and CD64 expression. For confirmation of provisional diagnosis of T and B ALL, after proper gating to isolate the lymphoblast population, further gating on blast cells that express TdT would help to differentiate patients with ALL, then confirming the nature of blasts as being T lymphoblasts by testing for cCD3 and B lymphoblasts by testing for CD10, CD22 and CD79a respectively. Results of FC-BMA and BMTB-immunohistochemistry are complementary to each other. A threshold of 10% MPO+ cells on either FC or BMTB would be sufficient to define the blast population/acute leukemia as belonging to the myeloid lineage, and a threshold of 5%, when values are ≥5% on both FC and BMTB.