Adult mesenchymal stem cells (MSCs) are promising tools for such applications as tissue engineering and cellular therapy. It is not clear how stem cells exposed to unfavorable conditions (e.g., hypoxia or inflammation) respond to signals of danger after in vivo transplantation. Toll-like receptors (TLRs) play a major role in the immune system, participating in the initial recognition of microbial pathogens and pathogen-associated components. This study was designated to determine the role of TLRs in human MSCs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry analysis demonstrated that MSCs derived from human adipose tissue and bone marrow express TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, and TLR-9. We investigated induction of the differentiation and proliferation of human adipose tissue stromal cells (hADSCs) by TLR agonists, including flagellin, peptidoglycans (PGN), lipopolysaccharide (LPS), the synthetic double-stranded RNA analog poly(I:C), and synthetic CpG oligodeoxydinucleotide (CpG-ODN). None of these agonists, except ODN, affected the proliferation of hADSCs. LPS and PGN increased osteogenic differentiation, but CpG-ODN decreased it. Poly(I:C) itself did not affect adipogenic or osteogenic differentiations, but exerted a synergistic effect on LPS- or PGN-induced osteogenic differentiation. RT-PCR analysis demonstrated that LPS and PGN induce osteogenic markers in hADSCs. TLR agonists affected the expression of chemokines and cytokines differentially. Furthermore, hADSCs affected the expression of specific TLRs in vitro under hypoxic conditions. These data provide evidence of a nonimmune role for TLR signaling on MSCs and may provide clues to the behavior of transplanted MSCs in vivo.