Allogenic chimeric antigen receptor (CAR)-transduced natural killer (NK) cells have garnered attention due to their efficacy, safety, and potential off-the-shelf nature in patients with refractory and relapsed hematological malignancies. However, producing clinical doses of genetically modified NK cells remains challenging. The current methods for ex vivo expansion and genetic modification of primary NK cells depend on irradiated feeder cells, particularly K562 leukemic cells enforced to express stimulatory molecules. However, this method is limited by high costs, difficulties in scaling up, licensing restrictions, and the potential risk of contamination of cancerous cells in the final cell products. Therefore, we aimed to develop a novel system to generate highly activated primary human NK cells using a combination of multiple cytokines and agonistic antibodies that stimulate CD2 and Natural Killer cell p46-related protein. We cultured unsorted peripheral blood mononuclear cells obtained from healthy adults ex vivo and selectively expanded primary NK cells using stimulatory antibodies and a combination of interleukin (IL)-2, IL-12, IL-18, and IL-21. The activated NK cells were successfully transduced using the RD114-pseudotyped retrovirus vector at high efficiency. Anti-CD19-BB-ζ CAR transduced NK cells showed strong cytotoxicity against B-cell tumors. The remarkable ability of this culture system to expand and generate CAR-NK cells will pave the way for allogenic cancer immunotherapy.