Abstract Natural killer (NK) cells are innate immune cells with the inherent ability to directly kill tumor and virus infected cells. Due to their ability to kill cancer cells without any prior priming, and their role in preventing metastasis NK cells have since long been the choice for autologous adoptive cell transfer therapy in cancer. However, poor expansion potential of PBMC derived NK cells in vitro is a major roadblock preventing the widespread use of NK cells in immunotherapy. We found that human NK cells isolated from lymph nodes (LNs) express higher levels of genes encoding for stem-like transcription factors (TCF7, LEF1, MYC) compared to NK cells from blood, spleen, bone marrow (BM) and lung. Therefore, we hypothesized that NK cells isolated from LNs will show superior expansion potential in vitro. Flow cytometric analysis shows that LN derived NK cells express high levels of TCF1 protein ex vivo, and show greater proliferation compared to NK cells isolated from blood, spleen and BM following stimulation with IL-2 and IL-15 in vitro. We also observed that a significant frequency of LN NK cells expressed TCF1 even after expansion, suggesting preserved proliferation potential of these cells. Additionally, the expanded NK cells from LN acquired properties of mature, highly functional NK cells such as increased expression of CD16, CD57 and higher Granzyme B expression. Lastly, LN derived NK cells also demonstrate enhanced cytolytic activity in vitro after expansion. Taken together our results suggest that in vitro expanded NK cells from LNs are potentially efficacious anti-tumor agents and could be leveraged for the development of future generation of NK cell directed immunotherapies.