The cell cycle regulatory proteins, which include cyclin-dependent kinases (cdks), cdk inhibitors (CKIs), cyclins, and the pRB, and E2F families of proteins, constitute a network of interacting factors which govern exit from or passage through the mammalian cell cycle. While the proteins within these families have similar structural characteristics, each family member exhibits distinct expression patterns during embryogenesis and distinct biological activities. In order to begin to understand the tissue-specific roles of these interacting factors, we determined the expression pattern and activity of the pRB, E2F, cyclin, cdk, and CKI families of cell cycle regulatory proteins during retinoic acid-induced (neuronal pathway) and DMSO-induced (cardiac muscle pathway) differentiation of the pluripotent murine embryonal carcinoma cell line, P19. We demonstrate here that P19 terminal differentiation causes lineage-specific changes in the expression and activity of distinct members of the E2F, pRB, cyclin, and CKI families. Furthermore, dynamic changes in the activities of these cell cycle regulatory proteins occur through several overlapping mechanisms, culminating in repression of DNA-binding activity by all of the E2F family members as cells terminally differentiate.