Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies. SYNOPSIS A barcoding‐based, high‐throughput approach enables multiplexed imaging of 3D spheroid microtissues. Quantitative single‐cell analyses show interdependence of global environment, local neighborhood, and internal cell state in determining cellular phenotype. A novel barcoding‐based, high‐throughput approach enables multiplexed mass cytometric imaging of 3D microtissues. A linear model quantifies environment, neighborhood, and internal cell state dependencies of marker expression. A strong interdependence is identified between environmental and internal cell state predictors of cellular marker expression. Systematic overexpression of signaling proteins within cells of 3D microtissues revealed non‐cell autonomous signaling. A barcoding‐based, high‐throughput approach enables multiplexed imaging of 3D spheroid microtissues. Quantitative single‐cell analyses show interdependence of global environment, local neighborhood, and internal cell state in determining cellular phenotype.