The mammalian Target of Rapamycin (mTOR) kinase functions within two structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2. The immunosuppressant and anticancer drug rapamycin is commonly used in basic research as a tool to study mTOR signaling. However, rapamycin inhibits only, and only incompletely, mTORC1, and no mTORC2-specific inhibitor is available. Hence, a full understanding of mTOR signaling in vivo, including the function of both complexes, requires genetic inhibition in addition to pharmacological inhibition. Taking advantage of the Cre/LoxP system, we generated inducible knockout mouse embryonic fibroblasts (MEFs) deficient for either the mTORC1-specific component raptor (iRapKO) or the mTORC2-specific component rictor (iRicKO). Inducibility of the knockout was important because mTOR complex components are essential. Induction of either raptor or rictor knockout eliminated raptor or rictor expression, respectively, and impaired the corresponding mTOR signaling branch. The described knockout MEFs are a valuable tool to study the full function of the two mTOR complexes individually.