Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B') but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila.