Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88^−/− and cot/tpl2^−/− mice. The matrix mineralization by the wild-type and cot/tpl2^−/− osteoblasts was significantly inhibited by LPS, whereas that of myd88^−/− was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88^−/− osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2^−/− osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the differentiation phase.