The organization of the genome in three-dimensional space is highly dynamic, yet how these dynamics are regulated and the role they play in genome function is poorly understood. Here we utilized acute depletion of NIPBL to characterize cohesin-mediated loop extrusion in vivo. We find that many chromatin loops are rapidly diminished upon loss of NIBPL, but some cohesin-dependent chromatin loops persist for multiple hours. These persistent loops required NIPBL for their establishment during mitotic exit, were associated with distinct chromatin states and were preferentially dependent on STAG1 for their persistence. Furthermore, by depleting NIPBL from multiple cell types, we find that NIPBL specifically regulates cell identity genes by supporting a unique local genome conformation defined by greater spatial proximity to nearby super-enhancers and weaker transcription start site insulation of genomic contacts. Overall, we show that NIPBL-mediated loop extrusion is critical to genome organization and transcription regulation in vivo.