T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.