Mouse lymphoid organs contain two major subsets of dendritic cells (DC) that differ in their phenotype and functions: conventional DC (cDC) and plasmacytoid DC (pDC). Recently, it has been proposed that differential expression of CCR9 could distinguish functionally distinct pDC subsets. We show that B220(+)CCR9(-) DC do not express classical pDC markers and have a developmental origin different from that of pDC. Furthermore, B220(+)CCR9(-) DC do not secrete IFN-alpha in response to CpG and, unlike pDC, can efficiently present exogenous Ags. Our results demonstrate that B220(+)CCR9(-) DC do not represent a subset of pDC. After in vivo transfer, these cells down-regulate B220 expression and convert into the two major cDC subsets, showing that they are a developmental stage of cDC differentiation.