The Hippo pathway, evolutionarily conserved from flies to mammals, promotes cell death and inhibits cell proliferation to regulate organ size. The core component of this cascade, Mst1 in mammalian cells, is sufficient to promote apoptosis. However, the mechanisms underlying both its activation and its ability to elicit cell death remain largely undefined. We here identify a signaling cassette in cardiac myocytes consisting of K-Ras, the scaffold RASSF1A, and Mst1 that is localized to mitochondria and promotes Mst1 activation in response to oxidative stress. Activated Mst1 phosphorylates Bcl-xL at Ser14, which resides in the BH4 domain, thereby antagonizing Bcl-xL-Bax binding. This, in turn, causes activation of Bax and subsequent mitochondria-mediated apoptotic death. Our findings demonstrate mitochondrial localization of Hippo signaling and identify Bcl-xL as a target that is directly modified to promote apoptosis.