Cells in vivo exist within the context of a multicellular tissue, where their behavior is governed by homo- and heterotypic cell-cell interactions, the material properties of the extracellular matrix, and the distribution of various soluble and physical factors. Most methods currently used to study and manipulate cellular behavior in vitro, however, sacrifice physiological relevance for experimental expediency. The fallacy of such approaches has been highlighted by the recent development and application of three-dimensional culture models to cell biology, which has revealed striking phenotypic differences in cell survival, migration, and differentiation in genetically identical cells simply by varying culture conditions. These perplexing findings beg the question of what constitutes a three-dimensional culture and why cells behave so differently in two- and three-dimensional culture formats. In the following review, we dissect the fundamental differences between two- and three-dimensional culture conditions. We begin by establishing a basic definition of what "three dimensions" means at different biological scales and discuss how dimensionality influences cell signaling across different length scales. We identify which three-dimensional features most potently influence intracellular signaling and distinguish between conserved biological principles that are maintained across culture conditions and cellular behaviors that are sensitive to microenvironmental context. Finally, we highlight state-of-the-art molecular tools amenable to the study of signaling in three dimensions under conditions that facilitate deconstruction of signaling in a more physiologically relevant manner.