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Background: The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle., Methods: We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types., Findings: Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells., Conclusions: Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases., Funding: This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132)., Competing Interests: Declaration of interests L.G.G. and V.H.G. have a patent application pending related to the hydrogel system., (Copyright © 2023 Elsevier Inc. All rights reserved.)