RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The RelbVenus/Venus mice developed without any abnormalities observed in the Relb–/– mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. RelbVenus/Venus DC analyses revealed that DCs consist of RelB–, RelBlow and RelBhigh populations. The RelBhigh population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelBlow population was in the cytoplasm. Although both the RelBlow and RelB– populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelBlow than in the RelB– splenic conventional DCs. Taken together, our results identify the RelBlow population as a possible novel intermediate maturation stage of cDCs and the RelbVenus/Venus mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.