While chemically defined conditions for culturing normal tissue have been attained for only a few cell types, the sustained proliferation of B precursor cells expressing IL-7 receptor and c-Kit can be supported under chemically defined conditions containing recombinant IL-7 and the ligand for c-Kit (KL). To understand the biochemical basis of the cell cycle progression of B precursor cells proliferating under these conditions, we investigated the correlation between growth factor stimulation and CDK4 activity. Consistent with our findings that IL-7 regulates the G1/S transition, while KL has only a little role in this process, the kinase activity of CDK4 was related closely with IL-7 stimulation but not KL stimulation. We investigated the mechanism underlying CDK4 activation in the IL-7 stimulated B precursor cells. Our results showed that (i) CDK4 and cyclin D3 are the G1/S regulators in B precursor cells; (ii) their expression levels are unchanged between the cells in G1 arrest and cycling cells; and (iii) they are present in an associated form even when the cell cycle stage is arrested at G1. Thus, the regulation of the expression of CDK4 and cyclin D3 or regulation of their assembly are not the mechanisms for activating CDK4 in the B precursor cells. On the other hand, a number of molecules co-immunoprecipitated with CDK4 were enhanced in the lysate of IL-7-stimulated B precursor cells. Thus, we present a possibility that CDK4 activation might be regulated by molecules associated with the CDK4-cyclin D3 complex in IL-7-dependent manner.