Natural Killer (NK) cells display potent cytotoxicity for cancer immunotherapy, influenced by cytokines like interleukin-2 (IL-2) that enhance their activation. Therapeutic IL-2 is widely used in NK cell immunotherapy, but high doses lead to severe side effects such as immunosuppression and vascular leakage syndrome. Therefore, it is crucial to develop NK cell lines with reduced dependency on IL-2. This research aims to create such cell lines through advanced cell engineering techniques, with a specific focus on manipulating cytoplasmic tyrosine kinases 1 and 3, which play a critical role in IL-2 signaling. We selected Janus kinase (JAK) activating mutations known to induce phosphorylation of downstream STAT5, which are dependent on IL-2. We cloned these mutations into expression vectors. Subsequently, we transfected NK cells to establish cell lines with sustained activation. Afterwards, when compared to the wild type, the engineered cell lines demonstrated over 20% higher cell growth rate at a low dose (10 IU/ml). Conducted various assays to assess the cytotoxicity of cell lines at low IL-2 concentrations. As a result, compared to the wild type (WT), all cell lines showed increased cytotoxicity. Among them, cell lines expressing JAK1 mutations (C clone), cell lines expressing JAK3 mutations (C clone), and cell lines expressing both JAK1 and JAK3 mutations (B clone) exhibited the highest cytotoxicity. In conclusion, the selected cell lines have exhibited reduced dependency on IL-2, enabling the maintenance of desired immune responses even at low IL-2 concentrations. This presents the potential to minimize the adverse effects that were previously associated with the use of high IL-2 dosages.