At the restriction point (R), mammalian cells irreversibly commit to division and will go on to divide even if growth factors are removed. R has mostly been viewed as a point in late G1 just before DNA replication when growth factors activate the kinase ERK to trigger a positive feedback loop of Cdk2 activity that makes progression through the cell cycle irreversible. However, recent single-cell studies cast doubt on this model by placing R much earlier in the cell cycle just after mitosis or even within the previous cell cycle. We developed a single cell assay to find that in primary cells passage through R occurs in G1 at the first passing of a threshold level of Cdk2 activity. While our data identify the threshold for R, we do not understand how growth factor signaling determines the rate of cell cycle progression through G1. Indeed, our preliminary data and reading of the literature suggests the presence of at least one additional, currently uncharacterized, control point (R0) in early G1. Current understanding of the impact of growth factor signaling on R is limited by the fact that only a handful of dynamic profiles of growth factors, such as a single ste increase or a single step decrease or pulse, have previously been investigated. To overcome technical limitations of low-throughput manual media exchange, we have created a microfluidics platform that integrates fluorescence imaging with sharp automated temporal control of the extracellular environment. We will use our microfluidics platform to examine cell cycle progression in mouse primary cells containing a variety of fluorescent reporters for cell cycle progression. This will allow us to test specific hypotheses for the molecular basis of R0 and the rate of progression through G1 to R. Specific Aims: (1) Use microfluidics to determine how primary cells respond to dynamic proliferation signals, (2) Determine how dynamic mitogen activated protein kinase (MAPK) signals control G1, and (3) Determine how dynamics of cell cycle inhibitors control G1. Cancer relevance: Our proposed systematic investigation of the proliferative response to dynamic growth factor signals will reveal distinct points of regulation within G1 beyond the previously characterized restriction point. This will give insight into normalproliferative control and its misregulation in cancer.