We have previously shown that TLR4 triggering promotes the generation of CD23(+)CD93(+) transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23(+) transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM(+)CD23(+)CD93(+) cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM(+)CD23(neg)CD93(+) cells. BAFF-R-Fc-mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-kappaB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM(+)CD23(+)CD93(+) cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23(+) B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.