Abstract NK cell differentiation mainly occurs in the bone marrow (BM) where a critical role in the regulation of developing lymphocyte distribution is played by members of the chemokine receptor family. In mouse, the chemokine receptor CX3CR1 identifies a late stage of NK cell development characterized by decreased effector functions and expression of the inhibitory receptor KLRG1. The role of CX3CR1 in the regulation of differentiation and positioning of NK cell subsets in the BM is not known. In this study, we found that CX3CR1 deficiency leads to accumulation of KLRG1+ NK cells in BM during steady-state conditions. The NK cell subset that expresses the receptor in wild-type mice was expanded in several tissues of CX3CR1-deficient mice, and NK cell degranulation in response to sensitive target cell stimulation was enhanced, suggesting a regulatory role of CX3CR1 in NK cell positioning and differentiation in BM. Indeed, the observed NK cell expansion was not due to altered turnover rate, whereas it was associated with preferential accumulation in the BM parenchyma. In addition, a role of CX3CR1 in NK cell trafficking from BM and spleen was evidenced also during inflammation, as CX3CR1-deficient NK cells were more prompt to exit the BM and did not decrease in spleen in response to polyinosinic-polycytidylic acid–promoted hepatitis. Overall, our results evidenced a relevant role of CX3CR1 in the regulation of NK cell subset exit from BM during homeostasis, and suggest that defect in the CX3CR1/CX3CL1 axis alters NK cell trafficking and functional response during inflammatory conditions.