BACKGROUND: In T-acute lymphoblastic leukemia (T-ALL), multi-parametric flow cytometry can serve to detect minimal residual disease (MRD) by using immature or aberrant antigens expression as well as the altered expression of T-cell antigens. The latter approach has been specifically introduced to overcome the absence of leukemia-associated antigens. However, there is no agreed-upon method for the use of T-cell antigens in T-ALL MRD testing. AIMS AND OBJECTIVES: To compare the expression of classic T-cell antigens on T-lymphoblasts and T-lymphocytes to establish a protocol for their use in MRD analysis. MATERIALS AND METHODS: Flow cytometric data of PB or BM samples from 63 adults with T-ALL were collected. We assessed the frequency and degree of brightness or dimness of each T-cell marker, in addition to studying the uniformity of the events scatter of a total of 287 follow-up BM samples from 50 patients. RESULTS: Significant differences in expression intensity of T-cell markers were found between T-lymphoblasts and T-lymphocytes; they were reasonably stable on blasts in follow up samples. This detailed study has nominated the conjoint use sCD3neg/dim and CD5dim/neg in the identification of residual cells, to be supported by other T-cell markers. CONCLUSION: The suggested gating sequence showed an acceptable level of accuracy in detecting residual leukemia, supporting their use in T-ALL MRD especially when other distinguishing markers might be absent in the diagnosis sample, or susceptible to be lost with induction therapy.