Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor xcexbaB (NF-xcexbaB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43xc2xb0C HS had an attenuated NF-xcexbaB p65 response to Tumor Necrosis Factor xcexb1 (TNFxcexb1) treatment. The degree of inhibition of the NF-xcexbaB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-xcexbaB responses while being consistent with the observed population-level responses. In particular all-or-nothing encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IxcexbaBxcexb1 phosphorylation, while others involving transport were analogue. In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-xcexbaB p65 subunit. The single cell responses exhibited all-or-nothing encoding. While most cells did not respond to TNFxcexb1 stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKxcexb1 and IKKxcexb2 subunits in whole cell lysates. However, a combination of all-or-nothing crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-xcexbaB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.