Simple SummaryThe kinetics of Cyclin Dependent Kinase 1 (CDK1) activation must be strictly controlled to guarantee a timely and physiological entry into mitosis. CDC6, a known S-phase regulator, has recently been found as a critical component in mitotic CDK1 activation cascade in early embryonic divisions. It acts due to association with Xic1 serving as abona fideCDK1 inhibitor upstream of Aurora A and Polo-Like Kinase 1 (PLK1), both of which are CDK1 activators. Here, we discuss the molecular processes that control the time of mitotic entry focusing on how the CDC6/Xic1 regulatory network affects CDK1 function. We present the existence of two distinct inhibitory mechanisms—Wee1/Myt1- and CDC6/Xic1-dependent—that slow down the kinetics of CDK1 activation, as well as how they interact with mechanisms that activate CDK1. The dynamics of CDK1 activation appear to be modulated by several inhibitors and activators, and their coordinated modulation ensures the robustness and some flexibility of mitotic timing. We therefore suggest a new model that incorporates CDC6/Xic1-dependent inhibition into the CDK1-activation cascade. This enables a better understanding of why cells divide at specific times and how the pathways involved in the timely regulation of cell division are all integrated to finely tune the control of mitotic events.AbstractTimely mitosis is critically important for early embryo development. It is regulated by the activity of the conserved protein kinase CDK1. The dynamics of CDK1 activation must be precisely controlled to assure physiologic and timely entry into mitosis. Recently, a known S-phase regulator CDC6 emerged as a key player in mitotic CDK1 activation cascade in early embryonic divisions, operating together with Xic1 as a CDK1 inhibitor upstream of the Aurora A and PLK1, both CDK1 activators. Herein, we review the molecular mechanisms that underlie the control of mitotic timing, with special emphasis on how CDC6/Xic1 function impacts CDK1 regulatory network in theXenopussystem. We focus on the presence of two independent mechanisms inhibiting the dynamics of CDK1 activation, namely Wee1/Myt1- and CDC6/Xic1-dependent, and how they cooperate with CDK1-activating mechanisms. As a result, we propose a comprehensive model integrating CDC6/Xic1-dependent inhibition into the CDK1-activation cascade. The physiological dynamics of CDK1 activation appear to be controlled by the system of multiple inhibitors and activators, and their integrated modulation ensures concomitantly both the robustness and certain flexibility of the control of this process. Identification of multiple activators and inhibitors of CDK1 upon M-phase entry allows for a better understanding of why cells divide at a specific time and how the pathways involved in the timely regulation of cell division are all integrated to precisely tune the control of mitotic events.