The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to study changes in cellular organization of human induced pluripotent stem cells (hiPSCs) as they differentiate into cardiomyocytes. We have used CRISPR/Cas9 to generate a collection of ∼35 endogenous fluorescently tagged hiPSC lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein that localizes to a particular cellular structure or organelle. In addition, we have developed several pertinent methods: 1) scarless GFP-tagging of late expressing cardiomyocyte genes, including ACTN2, ssTNNI1, MYL2, MYL7 and TTN, to study the organization and morphogenesis of the contractile apparatus; 2) a robust protocol for differentiation of hiPSCs into cardiomyocytes and methods for preparing cells for imaging; and 3) image-based assays and segmentation algorithms that enable single-cell analyses of structure localization in differentiated cardiomyocytes. Using the cardiomyocyte-specific protein ACNT2 as a reference, we have also developed methods for quantifying the extent of sarcomeric organization in cells across timepoints, thus placing individual cells along a psuedotime axis based on their level of structural ACTN2 organization. We are also using this approach to analyze sarcomere maturation via other key sarcomeric structural and regulatory proteins. This permits us to use sarcomere organization state as a reference system to analyze the reorganization of cytoplasmic structures. We are now incorporating multiplexed fluorescence in situ hybridization (FISH) into this analysis, allowing us to directly investigate the connection between cell structure organization and gene expression profiles on a single-cell level and also to produce structure-referenced maps of RNA localization. This imaging pipeline is generating an image database of high-resolution, high-replicate image data of the fluorescently tagged structures with a goal of generating an integrated image “state space” of intracellular reorganization during cardiomyocyte differentiation.