The Raf/MEK/ERK kinase cascade is pivotal in transmitting signals from membrane receptors to transcription factors that control gene expression culminating in the regulation of cell cycle progression. This cascade can prevent cell death through ERK2 and p90(Rsk) activation and phosphorylation of apoptotic and cell cycle regulatory proteins. The PI3K/Akt kinase cascade also controls apoptosis and can phosphorylate many apoptotic and cell cycle regulatory proteins. These pathways are interwoven as Akt can phosphorylate Raf and result in its inactivation, and Raf can be required for the antiapoptotic effects of Akt. In this study, the effects of activated Raf (Raf-1, A-Raf and B-Raf) and PI3K/Akt proteins on abrogation of cytokine dependence in FL5.12 hematopoietic cells were examined. Activated Raf, PI3K or Akt expression, by themselves, did not readily relieve cytokine dependence. The presence of activated Raf and PI3K/Akt increased the isolation of factor-independent cells from 400- to 2500-fold depending upon the particular combination examined. The individual effects of activated Raf and Akt on proliferation, apoptosis and autocrine growth factor synthesis were further examined with hormone-inducible constructs (Delta Raf-1:AR and Delta Akt:ER*(Myr(+)). Activation of either Raf or Akt hindered cell death; however, both proliferation and maximal synthesis of autocrine cytokines were dependent upon activation of both signaling pathways. The effects of small molecular weight inhibitors on DNA synthesis and cytokine gene expression were also examined. The PI3K inhibitor, LY294002, inhibited growth and cytokine gene expression. This effect could be synergistically increased by addition of the MEK inhibitor UO126. These cells will be useful in elucidating the interactions between Raf/MEK/ERK and PI3K/Akt cascades in proliferation, apoptosis, and leukemogenesis, as well as evaluating the efficacy of signal transduction inhibitors that target these cascades.