Human NK cells can be divided into CD56 dim CD16+ killer Ig-like receptors (KIR)+/− and CD56 bright CD16− KIR− subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56 bright NK cells mainly gain the signature of CD56 dim NK cells. Remarkably, KIR can be induced not only on CD56 bright, but also on CD56 dim KIR− NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56 dim display shorter telomeres than PB-and lymph node (LN)-derived CD56 bright NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56 bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR+ CD16+ NK cells. Altogether, our results suggest that CD56 bright CD16− KIR− and CD56 dim CD16+ KIR+/− NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.