Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)-like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cellsin vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF-κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF-κB signaling after CD40 stimulation and both B-cell receptor- and Toll-like receptor 9-mediated activation remained unaffected. Reduced canonical NF-κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression ofBcl-xLin MZ-like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL-derived B cells that were observedin vitro.The B-cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ-like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF-κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.