Mesenchymal stem cells (MSCs) are successful for their therapeutic application in immune and inflammatory contexts due to their anti-inflammatory, trophic, and immunomodulatory roles. However, though MSCs have the potential to provide regenerative treatment toward a wide range of devastating diseases, massive cell death of transplanted MSCs remains an obstacle to overcome. The relation between MSCs and inflammation is multifactorial and challenging to comprehend. Fever is a critical component of the inflamed microenvironment. Also, the choice of MSC source could be critical in determining the fate of transplanted cells under stress conditions. Here we investigated the thermosensitivity of Wharton's jelly MSCs (WJ-MSCs) to elevated temperature in the physiological fever range. We explored the effect of febrile range temperature on morphology, viability, proliferation kinetics, and cell cycle status of WJ-MSCs. WJ-MSCs adopted a flattened morphology at 40°C, and our data from proliferation kinetics study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that WJ-MSCs had reduced proliferation and viability at 40°C compared with control cultures. There was also a G0/G1 cell cycle arrest, which was further confirmed by messenger RNA (mRNA) levels of genes specific for different stages of cell cycle. On evaluating p53 status, we observed an increase in p53 protein expression and its nuclear localization in WJ-MSCs exposed to 40°C. Its downstream effector p21 too was upregulated. Moreover, this temperature-induced p53 induction was inhibited on exposure to 40°C in the presence of NF-κB pathway inhibitor, pyrrolidinedithiocarbamate (PDTC) or endonuclease-prepared small interfering RNA (esiRNA) targeting p65. Febrile temperature exposure did not affect the senescence status of WJ-MSCs. The MSC-specific surface antigen profile at 40°C was similar to control WJ-MSCs. Our findings suggest that under febrile temperature stress conditions, WJ-MSCs exhibit G0/G1 cell cycle arrest and reduction in viable cell count, while retaining their basic characteristics, with an underlying interplay of p53 and NF-κB pathway.