Background: T lymphocyte proliferation is central for adaptive immune response. Proliferation of T lymphocytes is initiated by the engagement of T cell receptor (TCR) and costimulatory receptors (CD28) which culminate with cell cycle entry and clonal expansion. This study aimed to use flow cytometry test to assay cell cycle of human T lymphocytes activated by anti-CD3 and anti-CD28 monoclonal antibodies in different incubation times in vitro. Methods: After obtaining 10 cc heparinized blood samples from normal volunteer donors, peripheral blood mononuclear cells (PBMCs) were separated by ficoll density-gradient centrifugation. The cells were then activated with anti human CD3 (OKT3) at 5 μg/ml and soluble anti human CD28 monoclonal antibody at 2 μg/mL in coated 24-well plates. Incubation was conducted at 37°C for 2 hours or at 4°C overnight. Finally, cell cycle analysis was performed using flow cytometry. Findings: Cell cycle histogram of activated T lymphocytes showed all the cells in G1 phase 24 and 48 hours after activation. However, activated cells entered into S and G2M phases 72 and 96 hours after activation. Conclusion: The degree and the length of TCR and CD28 occupancy are both critical for T cells to leave the G0 stage and enter different phases of cell cycle. Activation of Activated T cells need more than 24 and 48 hours to progress out of G1 phase. However, the cells could progress to S and G2 phases 72 and 96 hours after activation. Keywords: Cell cycle, T lymphocytes, Flow cytometry