AbstractDuring early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.