T cell acute lymphoblastic leukemia (T­ALL), a neoplasm derived from T cell lineage­committed lymphoblasts, is characterized by genetic alterations that result in activation of oncogenic transcription factors and the NOTCH1 pathway activation. The NOTCH is a transmembrane receptor protein activated by γ­secretase. γ­secretase inhibitors (GSIs) are a NOTCH­targeted therapy for T­ALL. However, their clinical application has not been successful due to adverse events (primarily gastrointestinal toxicity), limited efficacy, and drug resistance caused by several mechanisms, including activation of the AKT/mTOR pathway. Nelfinavir is an human immunodeficiency virus 1 aspartic protease inhibitor and has been repurposed as an anticancer drug. It acts by inducing endoplasmic reticulum (ER) stress and inhibiting the AKT/mTOR pathway. Thus, it was hypothesized that nelfinavir might inhibit the NOTCH pathway via γ­secretase inhibition and blockade of aspartic protease presenilin, which would make nelfinavir effective against NOTCH­associated T­ALL. The present study assessed the efficacy of nelfinavir against T­ALL cells and investigated mechanisms of action in vitro and in preclinical treatment studies using a SCL­LMO1 transgenic mouse model. Nelfinavir blocks presenilin 1 processing and inhibits γ­secretase activity as well as the NOTCH1 pathway, thus suppressing T­ALL cell viability. Additionally, microarray analysis of nelfinavir­treated T­ALL cells showed that nelfinavir upregulated mRNA levels of CHAC1 (glutathione­specific γ­glutamylcyclotransferase 1, a negative regulator of NOTCH) and sestrin 2 (SESN2; a negative regulator of mTOR). As both factors are upregulated by ER stress, this confirmed that nelfinavir induced ER stress in T­ALL cells. Moreover, nelfinavir suppressed NOTCH1 mRNA expression in microarray analyses. These findings suggest that nelfinavir inhibited the NOTCH1 pathway by downregulating NOTCH1 mRNA expression, upregulating CHAC1 and suppressing γ­secretase via presenilin 1 inhibition and the mTOR pathway by upregulating SESN2 via ER stress induction. Further, nelfinavir exhibited therapeutic efficacy against T­ALL in an SCL­LMO1 transgenic mouse model. Collectively, these findings highlight the potential of nelfinavir as a novel therapeutic candidate for treatment of patients with T­ALL.