Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well as caspase-3,-8, and-9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B into the cytosol, nuclear condensation, and processing of caspase-8 and-3. According to our model, activation of caspase-3 by caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the apoptotic program induced by GCs comprises cell type-specific as well as common features.