The duration and distance of manned space flights emphasizes the importance of advanced elucidation of space flight factors and their effects on human beings. The exposure to inflammatory mediators under microgravity may contribute to the activity of different cells, perivascular stromal cells (MSCs) in particular. Inflammatory activation is now considered as a principal cue of MSC engagement in reparative remodeling. In the present paper, the effect of simulated microgravity (sµg) on TNFα-mediated priming of adipose tissue-derived MSC (ASCs) was examined. Sµg per se did not induce inflammatory-related changes, such as elevation of ICAM-1 and HLA-ABC expression, soluble mediator production, or shifting of the transcription profile in ASCs. Moreover, the attenuated ASC response to TNFα priming under sµg was manifested in decreased production of TNFα-dependent pleiotropic cytokines (IL-8 and MCP-1), matrix remodeling proteases, and downregulation of some genes encoding growth factors and cytokines. Time-dependent analysis detected the first signs of priming attenuation after 48 hours of 3D-clinorotation. A reduced response of MSCs to priming under sµg can be a negative factor in terms of MSC involvement in tissue remodeling processes.