Ewing Sarcoma (ES) shows several deregulated autocrine loops mediating cell survival and proliferation. Therefore, their blockade is a promising therapeutic approach. We previously reported the in vitro effect of insulin-like growth factor 1 receptor (IGF1R)/KIT pathway blockade on ES cell lines, and we now extend our observations to changes induced by this treatment in interacting proteins/networks. A proteomic analysis revealed that Heat Shock Protein (HSP)90 was differentially expressed between ES cell lines sensitive and resistant to specific IGF1R/KIT inhibitors. We therefore inhibited HSP90 with 17-allylamino-17-demethoxygeldanamycin (17-AAG) and siRNA, and observed that ES cell line growth and survival were reduced, especially in the resistant cell lines. Conversely, HSP90 induced–expression conferred resistance to anti-IGF1R/KIT treatment in the sensitive cell lines. 17-AAG treatment induced HSP90 client protein degradation, including AKT, KIT, or IGF1R, by inhibiting their physical interaction with HSP90. Xenograft models developed with A673 ES cell line confirmed that HSP90 inhibition, alone or combined with IGF1R inhibition, significantly reduced tumor growth and expression of client proteins. Remarkably, using two independent clinical sample sets, we have found that nearly half of IGF1R-positive tumors also show HSP90 overexpression. This delineates a subset of patients that could benefit from combination of anti-HSP90 agents when considering IGF1R-targeting therapies. Importantly, sensitivity to drugs such as ADW/IMA depends not only on the levels of expression and basal activation of IGF1R/KIT, but also, and for the first time reported in ES, on the development of the stress response mechanism. Accordingly, HSP90 expression could be a predictive factor of response to IGF1R-targeting therapies. [Cancer Res 2008;68(15):6260–70]