NK cells are initially known for their ability to kill tumor cells with no prior sensitization. Production of mature and long lasting NK cells from Umbilical Cord Blood (UCB) by using cytokines could be a promising method for immunotherapy. NK cells were generated from cord blood cells using IL2, IL7, and IL15 cytokines and measured expression of CD57 and NKp46 markers. Afterward, their capacity in the elimination of malignant cells (Reh cell line) was evaluated by assessment of interferon-γ (as cytokine production sign) and CD107-a expression (as cytotoxic function symptom) using flow cytometry. Our results showed efficient NKp46 + , and CD57 + NK cells generated on day 14. Also, expression of CD107-a and IFN-γ following co-culture with Reh cell lines significantly increased in comparison to the control. Taken together, we have reported one of the best culture conditions for the generation of CD57 + NK cells with on feeder cells and showed appropriate capacity in counter reh cell lines as a target.