Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.