Mesenchymal stem cells (MSCs) can be polarized into two distinct populations, MSC1 and MSC2, by activation of different Toll-like receptors (TLRs). TLR4-primed MSC1 expressed proinflammatory factors, whereas TLR3-primed MSC2 expressed suppressive factors. However, little is known about the function of TLRs on B lymphocyte-related immune modulation. In this study, we investigated the expression of B cell activating factor (BAFF), a member of the tumor necrosis factor ligand superfamily with notable stimulating activity on B cells, in human MSCs (hMSCs) and in murine MSCs (mMSCs) after activation of TLRs. BAFF was increasingly expressed in presence of TLR4 agonist (lipopolysaccharide, LPS), while TLR2 agonist (Zymosan) and TLR3-agonist (polyinocinic-polycytidykic acid, poly I:C) had no effect on BAFF expression. In addition, we demonstrated that signaling pathways of NF-κB, p38 MAPK, and JNK were involved in TLR4-primed BAFF expression. Our results suggested that TLR4 and downstream pathways in MSCs exert an important function in B lymphocyte-related immune regulation. Further defining a homogeneous population of MSCs should provide insight into MSC-based immune-modulating therapy.