Phosphatidylinositol (PI) 3-kinase mediates multiple pathways that regulate many aspects of the cell including metabolism, survival, migration, and proliferation. Both Akt and cytokine-independent survival kinase (CISK)/SGK3 are known AGC family protein kinases that function downstream of PI 3-kinase. Although the Akt signaling pathway has been studied extensively, the specific signaling cascades that are modulated by CISK remain to be elucidated. To understand CISK function, we affinity-purified the CISK protein complex and identified Flightless-I (FLII) as a novel downstream target of CISK. Here we show that FLII is an in vivo substrate of CISK that functions downstream of PI 3-kinase. CISK can associate with FLII and phosphorylate FLII at residues Ser(436) and Thr(818). FLII has been shown to act as a co-activator for nuclear hormone receptors such as estrogen receptor (ER). We demonstrate here that CISK can enhance ER transcription, which is dependent on its kinase activity, and mutation of CISK phosphorylation sites on FLII attenuates its activity as an ER co-activator. Furthermore, FLII knockdown by RNA interference renders 32D cells more sensitive to interleukin-3 withdrawal-induced apoptosis, suggesting that FLII itself is also a survival factor. These findings support the model that CISK phosphorylates FLII and activates nuclear receptor transcription and suggest a new cell survival signaling pathway mediated by PI 3-kinase and CISK.