Laboratory and epidemiological studies have provided indirect but compelling evidence that toll-like receptor (TLR) signaling pathways play an important role in host responsiveness to ambient immunostimulatory factors. Nonetheless, direct evidence is limited. This paper will present our experience investigating the innate immunostimulatory activities of sterile house dust extracts (HDEs). In initial studies, bone marrow derived dendritic cells (BMDDCs) were cultured with HDEs, and cytokine production and co-stimulatory molecule expression were evaluated. In additional experiments, the TLR dependence of these responses was determined. HDEs induced concentration-dependent BMDDC activation. Moreover, the relative bioactivities of HDEs correlated with their endotoxin content. Finally, HDE-mediated responses were found to be partially dependent on TLR2, TLR4, and TLR9 and almost completely dependent on MyD88. These investigations provide the first direct evidence that TLR signaling pathways play a key role in innate responsiveness to non-infectious factors ubiquitous in living environments.