ABSTRACTNatural killer (NK) cells, integral to the innate immune system, are notable in cell therapies because of their applicability in allogeneic treatments, distinguishing them from T cells typically employed in conventional cell therapies. However, their limited half‐life (proliferative capability) poses a challenge for therapy. The limited half‐life creates difficulties in obtaining a sufficient number of cells for in vitro adoptive therapy. Gene modification is commonly employed to address this limitation. However, due to concerns such as genetic instability and unintended gene expression, its suitability for long‐term cultivation is uncertain. Consequently, safer alternatives are needed. We aimed to promote NK cell proliferation through feeder cells rather than genetic modification. These cells are designed to interact with NK cells without adverse effects, aiming to promote NK cell proliferation more safely. In our study, during the tailoring of feeder cells, we excluded genetic modification and instead applied chemical‐based extracellular stress. The extracellular stress applied consisted of hypoxia and cytochalasin D. By treating the feeder cells with these stressors, we were able to inhibit feeder cell proliferation, enabling them to function more efficiently as feeder cells. Furthermore, we observed that the feeder cells subjected to extracellular stress exhibited upregulated expression of 4‐1BBL, which enhances the 4‐1BB/4‐1BBL interaction with NK cells. The upregulated 4‐1BBL binds to 4‐1BB on the surface of NK cells, promoting their proliferation. Additionally, following coculture with feeder cells exposed to extracellular stress, we observed an upregulation of CD56 expression on the surface of NK cells. These CD56bright NK cells influence NK cell proliferation through enhanced cytokine release. We further validated this process under dynamic conditions where shear stress is applied, demonstrating that the feeder cell‐mediated enhancement of NK cell proliferation is applicable under dynamic conditions such as those found in bioreactors.