The genome-wide set of Saccharomyces cerevisiae deletion strains provides the opportunity to analyze how other organisms may respond to toxic agents. Since arsenic trioxide selectively kills human acute promyelocytic leukemia (APL) cells by a poorly understood mechanism we screened the yeast deletion strains for sensitivity or resistance. In addition to confirming mutants previously identified as sensitive to sodium arsenite, a large number of additional genes, and cellular processes, were required for arsenic trioxide tolerance. Of the 4546 mutants, 7.6% were more sensitive to arsenic trioxide than the wild type, while 1.5% was more resistant. IC50 values for all sensitive and resistant mutants were determined. Prominent as sensitive was that missing the MAP kinase, Hog1. The most resistant lacked the plasma-membrane glycerol and arsenite transporter, Fps1. Hog1 and Fps1 control the response to osmotic stress in yeast by regulating glycerol production and plasma membrane flux, respectively. We therefore tested whether APL cells have impaired osmoregulation. The APL cell line NB4 did not produce glycerol in response to osmotic stress and underwent apoptotic cell death. Moreover, the glycerol content of NB4 and differentiated NB4 cells correlated with the level of arsenic trioxide uptake and the sensitivity of the cells. Additionally, NB4 cells accumulated more arsenic trioxide than non-APL cells and were more sensitive. These findings demonstrate the usefulness of the S. cerevisiae deletion set and show that the selectivity of arsenic trioxide for APL cells relates, at least in part, to impaired osmoregulation and control of uptake of the drug.