Deregulation of D-type cyclin-dependent kinases (CDK4 and 6) is widely observed in various human cancers, illustrating their importance in cell cycle control. Like other cyclin-dependent kinases (CDKs), assembly with cyclins is the most critical step for activation of CDK4/6. As previously reported elsewhere, we observed that the level of cyclinD1-CDK4 complex and its associated kinase activity were significantly low in asynchronously proliferating mouse embryo fibroblasts lacking both p21(Cip1) and p27(Kip1) (p21/p27-null MEFs). These evidences imply that p21(Cip1) and p27(Kip1) CDK inhibitors are 'essential activators' of cyclin D-kinases. We, however, discovered here that both the assembly and activation of cyclin D1-CDK4 complex occur when quiescent p21/p27-null MEFs were stimulated to re-enter the cell cycle. This mitogen-induced cyclin D1-kinase activity was blocked by overexpression of p16(INK4a) and resulted in the inhibition of S phase entry in p21/p27-null MEFs. Furthermore, ectopic expression of p34(SEI-1), a mitogen-induced CDK4 binding protein, increased the levels of active cyclinD1-CDK4 complex in asynchronously proliferating p21/p27-null MEFs. Together, our results suggest that there are several independent ways to stimulate the assembly of cyclin D1-CDK4 kinases. Although p21(Cip1) and p27(Kip1) play a role in this process, our results demonstrate that additional mechanisms must occur in G0 to S phase transition.