The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in tumorigenesis because it affects the growth and survival of cancer cells. Several laboratories have demonstrated that Akt inhibits transcriptional activation of a number of related forkhead transcription factors now referred to as FoxO1, FoxO3, and FoxO4. Akt-regulated forkhead transcription factors are involved in the control of the expression of both the cyclin-dependent kinase (cdk) inhibitor p27(Kip1) and proapoptotic Bim protein. Very little information is available concerning the importance of the PI3K/Akt pathway in HL60 human leukemia cells. Here, we present our findings showing that the PI3K/Akt axis regulates cell cycle progression of HL60 cells through multiple mechanisms also involving the control of FoxO1 and FoxO3. To this end, we took advantage of a HL60 cell clone (HL60AR cells) with a constitutively activated PI3K/Akt axis. When compared with parental (PT) HL60 cells, HL60AR cells displayed higher levels of phosphorylated FoxO1 and FoxO3. In AR cells forkhead factors localized predominantly in the cytoplasm, whereas in PT cells they were mostly nuclear. AR cells proliferated faster than PT cells and showed a lower amount of the cdk inhibitor p27(Kip1), which was mainly found in the cytoplasm and was hyperphosphorylated on threonine residues. AR cells also displayed higher levels of cyclin D1 and phosphorylated p110 Retinoblastoma protein. The protein levels of cdk2, cdk4, and cdk6 were not altered in HL60AR cells, whereas the activities of both ckd2 and cdk6 were higher in AR than in PT cells. These results show that in HL60 cells the PI3K/Akt signaling pathway may be involved in the control of the cell cycle progression most likely through mechanisms involving the activation of forkhead transcription factors.