The human placenta is comprised of trophoblast subtypes – cytotrophoblasts, syncytiotrophoblasts (ST), and extravillous trophoblasts (EVT) – that are crucial for successful pregnancies. Understanding trophoblast functions is essential to treat pregnancy complications and investigate maternal-fetal immune tolerance. Trophoblasts secrete factors to instruct tolerance; however, the distinct trophoblast subtypes’ role in fetal tissue tolerance remain insufficiently understood. Inadequate models to study the human placenta in vitro limit the current understanding of human placental behavior and development. Synthetic hydrogel systems such as poly(ethylene) glycol (PEG)-maleimide offer a highly defined, tunable 3D environment to study trophoblast subtypes, which may overcome experimental variability in naturally derived hydrogels like Matrigel due to batch-to-batch variability. Here, a PEG hydrogel system with tunable degradability and placenta-derived extracellular matrix ligands is employed to evaluate the capacity of the hydrogel library to support the function and phenotypic protein expression of three trophoblast-like cell lines, assess the differentiation of trophoblast stem cells (TSC), and explore the effects of trophoblast supernatants on the modulation of protein expression and secretion by immune cell subsets present at the maternal-fetal interface. Degradable synthetic hydrogels support the greatest degree of trophoblast-like spheroid proliferation and function relative to standard Matrigel controls and culture conditions modulate trophoblast-like and TSC functional subtype as measured by proteomics analysis and functional secretion assays. PEG hydrogels support TSC viability and function comparable to Matrigel; however, ST-differentiated cells prefer PEG environments, while EVT-differentiated cells favor Matrigel, as assessed through phenotypic protein expression and secretion. These data highlight the influence of trophoblast culture condition and subtype on immune cell protein expression and inflammatory cytokine secretion in response to trophoblast supernatants. This model advances the understanding of in vitro placental modeling, which can provide insights on pregnancy-related complications and maternal-fetal immunotolerance.