Loss of Human Leukocyte Antigen (HLA) class I molecules can occur in infection and cancer, and be detected by Natural Killer (NK) cells via inhibitory Killer cell Immunoglobulin-like Receptors (KIR). However, while the function of NK cells is largely predicated on the ‘missing self’ hypothesis, where HLA absence prevents inhibitory signalling and allows NK cell activation, HLA loss is not always complete, can be allotype specific and, notably during infection, can occur over time. Furthermore, the vast polymorphism of HLA and KIR across the population yields pairings of varying affinity, which has the capacity to differentially impact NK cell function between individuals. Assessment of the binding strength of KIR/HLA pairs using tetrameric KIR3DL1 molecules and HLA-coated beads identified allotype-specific differences in binding. While some allotypes like HLA-B*57:01 strongly bound to all KIR3DL1 allotypes, others such as HLA-A*24:02 differentially bound to particular KIR3DL1 allotypes. The capacity of KIR3DL1+ NK cells from donors of known KIR3DL1/HLA type to respond to HLA-deficient targets correlated with the strength of their KIR3DL1/HLA pairs, implicating this as a primary parameter driving education. However, when encountering targets with reduced HLA levels, as induced by viral evasins, an inverse relationship was observed where weaker KIR3DL1/HLA interactions allowed more robust responses. Moreover, when HLA loss was examined over time in reporter cell assays, stronger KIR/HLA pairs retained HLA recognition longer than those with weaker pairings. This suggests that while high affinity KIR/HLA interactions potently educate NK cells, lower affinity interactions may increase their capacity to sense more subtle changes in HLA expression. Therefore, disease outcomes may vary depending on the extent or nature of HLA loss, with NK cells educated by high affinity KIR/HLA pairings being beneficial in situations of total HLA loss and weaker HLA/KIR pairs more effective when HLA loss is gradual or only partial.