Multipotent mesenchymal stromal cells (MSCs) are considered cue regulators of tissue remodelling. Their activity is strongly governed by local milieu, where O2 level is most important. The elevation of inflammatory mediators and acute O2 lowering may additionally modulate MSC activity. In present paper the priming effects of IFN‐gamma on adipose tissue‐derived MSCs (ASCs) at tissue‐related O2 level (5%) and acute hypoxic stress (0.1% O2) were assessed as alterations of ASCs' CFU‐F, proliferation, migration, osteo‐commitment. IFN‐gamma priming provoked ROS elevation, cell growth slowdown, attenuation of both spontaneous and induced osteodifferentiation of tissue O2‐adapted ASCs. The prominent changes in ASC cytoskeleton‐related gene transcription was detected. IFN‐gamma exposure shifted the ASC paracrine profile, suppressing the production of VEGF and IL‐8, while MCP‐1 and IL‐6 were stimulated. Conditioned medium of IFN‐gamma‐primed ASCs did not activate vessel growth in the CAM assay, but induced endothelial cell migration in “wound closure”. Short‐term hypoxia suppressed CFU‐F number, IFN‐gamma‐induced elevation of IL‐6 and endothelial cell migration, while it abolished IFN‐gamma‐provoked VEGF inhibition. After N‐acetyl cysteine treatment ROS level was partly abolished providing additional enhancement of IL‐6 and suppression of IL‐8 and VEGF production. These findings demonstrated that paracrine activity of ASCs in part may be governed by ROS level. Thus, this study first demonstrated that IFN‐gamma priming itself and in combination with acute O2 deprivation could supply dual effects on ASC functions providing both stimulatory and hampering effects. The equilibrium of these factors is a substantial requirement for the execution of MSC remodelling functions. This article is protected by copyright. All rights reserved