The incorporation of the extracellular matrix (ECM) is essential for generating in vitro models that truly represent the microarchitecture found in human tissues. However, the cell-cell and cell-ECM interactions in vitro remains poorly understood in placental trophoblast biology. We investigated the effects of varying the surface properties (surface thickness and stiffness) of two ECMs, collagen I and Matrigel, on placental trophoblast cell morphology, viability, proliferation, and expression of markers involved in differentiation/syncytial fusion. Most notably, thicker Matrigel surfaces were found to induce the self-assembly of trophoblast cells into 3D spheroids that exhibited thickness-dependent changes in viability, proliferation, syncytial fusion, and gene expression profiles compared to two-dimensional cultures. Changes in F-actin organization, cell spread morphologies, and integrin and matrix metalloproteinase gene expression profiles, further reveal that the response to surface thickness may be mediated in part through cellular stiffness-sensing mechanisms. Our derivation of self-assembling trophoblast spheroid cultures through regulation of ECM surface alone contributes to a deeper understanding of cell-ECM interactions, and may be important for the advancement of in vitro platforms for research or diagnostics.