We evaluated the effect of beta1-integrin receptor engagement on the expression and activity of cell cycle regulatory proteins in CD34(+) cells under conditions that mimic the steady-state marrow microenvironment and in the presence of supraphysiological concentrations of interleukin-3 (IL3) and stem cell factor (SCF). Adhesion of CD34(+) progenitors to fibronectin (FN) was similar whether IL3 or SCF was present or absent. Engagement of beta1-integrins blocked S-phase entry of CD34(+) cells in the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF prevented such a block in S-phase entry. In the absence of IL3 or SCF, cyclin-E levels were significantly lower and p27(KIP1) levels significantly higher in FN-adherent than in FN-nonadherent cells, or than in poly-L-lysine (PLL)-adherent or (PLL)-nonadherent cells. Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of cyclin-E-cdk2 complexes were lower in FN-adherent than in PLL-adherent cells. In contrast, cyclin-E and p27(KIP1) protein levels and cdk2 activity in cells adherent to FN in the presence of IL3 or SCF were similar to those in PLL-adherent and FN-nonadherent or PLL-nonadherent cells. In conclusion, under physiological cytokine conditions, integrin engagement prevents S-phase entrance of CD34(+) cells, which is associated with elevated levels of the contact-dependent cyclin kinase inhibitor p27(KIP1). Supraphysiological concentrations of IL3 or SCF prevent p27(KIP1) elevation and override the integrin-mediated inhibition of entry into S phase.