Pluripotent stem cell differentiation recapitulates aspects of embryonic development, including the regulation of morphogenesis and cell specification via precise spatiotemporal signaling. The assembly and reorganization of cadherins within multicellular aggregates may similarly influence β-catenin signaling dynamics and the associated cardiomyogenic differentiation of pluripotent embryonic stem cells (ESCs). In this study, dynamic changes in β-catenin expression and transcriptional activity were analyzed in response to altered cell adhesion kinetics during embryoid body (EB) formation and differentiation. Modulation of intercellular adhesion kinetics by rotary orbital mixing conditions led to temporal modulation of T-cell factor/lymphoid enhancer-binding factor activity, as well as changes in the spatial localization and phosphorylation state of β-catenin expression. Slower rotary speeds, which promoted accelerated ESC aggregation, resulted in the early accumulation of nuclear dephosphorylated β-catenin, which was followed by a decrease in β-catenin transcriptional activity and an increase in the gene expression of Wnt inhibitors such as Dkk-1. In addition, EBs that exhibited increased β-catenin transcriptional activity at early stages of differentiation subsequently demonstrated increased expression of genes related to cardiomyogenic phenotypes, and inhibition of the Wnt pathway during the initial 4 days of differentiation significantly decreased cardiomyogenic gene expression. Together, the results of this study indicate that the expression and transcriptional activity of β-catenin are temporally regulated by multicellular aggregation kinetics of pluripotent ESCs and influence mesoderm and cardiomyocyte differentiation.