NK cells co-expressing NKG2C and inhibitory KIRs represent promising effector candidates when directed against HLA-E tumors. These cells may propagate in vitro upon HCMV peptide (LFL) presentation within HLA-E by interacting cells due to KIRNKG2C cell expansion or via NKG2C or KIR de novo expression. This study aimed to clarify whether these accumulation mechanisms are connected to the healthy individual's ex vivo NK cell phenotype. The architecture of the phenotype was characterized by the distribution of markers known to be associated with NK cell differentiation and the HCMV adaptive response, including NKG2C, KIR2DL2/3, and HLA-DR. We also analyzed NK cells that differed in marker expression cultured with or without LFL in vitro. The most pronounced NKG2C expression de novo was observed in KIR NK cells, associated with a higher HLA-DR ex vivo proportion. Enhanced LFL-dependent KIRNKG2C cell expansion of the CD57 fraction was associated with a high content of adaptive NK cells ex vivo, while the CD57KIRNKG2C expansion rate correlated with a high level of NKG2A ex vivo. Moreover, LFL-activated cultures more effectively eliminated HLA-E tumor spheroids. These data are of interest both for KIRNKG2C NK cell therapeutic application and for widening understanding of virus-induced NK cell repertoire modulation.