To improve our understanding of human placental function and placental cell responses to pregnancy stressors, the development of in vitro models that better recapitulate the in vivo placental microenvironment is needed. Here, we describe a three-dimensional (3D) silicone polymer polydimethylsiloxane (PDMS) microfluidic platform for modeling human trophoblast invasion recreating a placental heterocellular microenvironment. This platform allows the formation of a cellular barrier establishing a chemical gradient and real-time evaluation of trophoblast cell invasion and heterocellular cell-to-cell interactions.