Abstract Body: Following myocardial infarction, the adult human heart undergoes pathological remodeling, developing noncontractile scars that impair cardiac function. In contrast, the neonatal mouse heart can efficiently regenerate damaged tissue; however, the mechanisms that coordinate regenerative responses across diverse cardiac cell types within the injury milieu remain poorly understood. Here, we combine spatial transcriptomics, single-cell analysis, and functional perturbation to define cell–cell communication programs underlying neonatal heart regeneration. Spatial profiling of regenerative neonatal and non-regenerative later-stage mouse hearts following ischemic injury revealed distinct injury trajectories across infarct, border, and remote tissue. In particular, the injury border zone tissue emerged as a critical site of divergence, where regenerative hearts uniquely activate transient stress responses along with activation of glycolysis and translation, leading to increased regenerative cardiomyocyte state over time. To identify microenvironmental drivers of the distinct border zone response, we developed Single-Cell REgenerative Environmental Mapping by sequencing (SCREEM-seq), a method that assesses the effect of signaling ligands on a cardiac tissue-derived in vitro cell ecosystem. Integrating transcriptome-based machine learning with functional validation, we found that ligands enriched in the neonatal regenerative microenvironment predominantly induce cell type–specific programs, despite their receptors are broadly expressed. Specifically, we identified OSM, IL-17A, and CCL24, secreted from neonatal immune cells, as key factors promoting regenerative responses in cardiomyocytes, fibroblasts, and endothelial cells, respectively. We show that combined delivery of these factors improved cardiac repair in non-regenerative mice. Together, these findings define spatially organized regenerative programs and identify microenvironmental signals that coordinate cell type–specific repair, providing a framework for therapeutic regeneration.