Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting the critical parameters associated with FACS methodologies have complicated interpretation, comparison, and reproduction of findings. To address this, a comprehensive multi-center study was designed to develop guidelines that limit experimental and data reporting variability, providing a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and post-sort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh1) and differentiated cell lineages (Lysozyme, Mucin2, ChromograninA and Sucrase Iso-maltase) were interrogated in target and control populations to assess intra- and inter-center variability. Wilcoxon rank sum test on gene expression levels showed limited intra-center variability between biological replicates. Principle component analysis demonstrated significant inter-center reproducibility among four centers. Analysis of data collected using standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post-hoc error identification. These results indicate the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.