The experiments described in this report were designed to determine if suppression of tumorigenicity can be mediated by cell-cycle-dependent mechanisms that control cellular differentiation and/or proliferation in mesenchymal stem cells of the 3T3 T type. These cells were employed because they possess distinct, well-characterized cell-cycle-dependent mechanisms to control both cellular differentiation and proliferation. To achieve our goal we developed by non-mutagenic procedures 23 clonal variants of 3T3 T stem cells that expressed one of 4 distinct phenotypes for the regulation of cellular differentiation and proliferation. Six clones expressed combined defects in the control of differentiation and proliferation; 6 expressed intact mechanisms t o control proliferation but defects in the control of differentiation; and 3 expressed intact mechanisms to control differentiation but defects in the control of proliferation. Finally, 8 clones expressed no detectable phenotypic defects in the control of either differentiation or proliferation. Once isolated and characterized, each of these clones was assayed for i t s tumorigenic potential. The results establish that clones which express combined defects in the control of differentiation and proliferation are highly tumorigenic. By contrast, tumorigenicity is markedly suppressed in clones that maintain the ability to control their proliferation or their differentiation. Furthermore, clones that maintained the ability to control both proliferation and differentiation showed no evidence of tumorigenicity. These data are interpreted t o suggest that stringently regulated control of cellular differentiation and/or proliferation can act as a cancer suppressor mechanism.