Multiple receptors have been implicated in the activation of Natural Killer (NK) cells, with some capable of independently driving NK cell responses and others cooperating to enhance activation. However, the mechanisms underlying these synergistic interactions remain poorly understood. When the expression of the ligands for the co-activating receptor DNAM-1 were manipulated on 221 and 293T cells, IFN-γ production by NK cells was found to be altered, while degranulation (CD107a) and CCL4 production were less affected. To further explore the role of DNAM-1 co-engagement, we crosslinked DNAM-1, 2B4, and NKG2D using monoclonal antibodies and assessed NK cell responses, including degranulation, chemokine, and cytokine production. While CCL4 levels remained unaffected, combinations of DNAM-1+2B4 and NKG2D+2B4+DNAM-1 induced enhanced degranulation and notably increased IFN-γ production. RNA-seq analysis showed that co-stimulation with DNAM-1+2B4 upregulated 33 genes, including IFN-γ and epigenetic regulators, beyond the levels observed with individual receptor engagement, highlighting their synergistic effects. Interestingly, co-crosslinking the inhibitory receptor TIGIT only modestly inhibited NK cell activation, with minimal changes in gene expression confirmed by RNA-seq. Further investigation into DNAM-1 and 2B4 synergism using CRISPR-edited and/or transfected 221 and K562 cell lines revealed a context-dependent interaction. IFN-γ production was significantly enhanced by the addition of CD155 to 221 cells, which express CD48, while in K562 cells, the addition of CD48 to CD155-expressing cells enhanced IFN-γ production independently of DNAM-1 signalling. This suggests that the synergy between DNAM-1 and 2B4 is modulated by the presence of other receptor-ligand interactions. Taken together, our data reveal that NK cell responses can be differentially regulated based on receptor co-engagement patterns and their specific ligands, underscoring the potential targeting of receptor interactions to optimize NK cell-based therapies.