Micropatterning techniques have enabled the use of 3D cell cultures to recreate tissue-level behavior such as hypoxia or signaling gradients, but their integration with microfluidics has been limited. To access complex, non-linear and concentric geometries seen in vivo and in high-throughput culture arrays, we developed an in-situ micropatterning strategy that integrated photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing. By shining 405 nm light through a photomask containing the desired design, we patterned 3D cultures directly in a 130-μm deep microfluidic chamber. As a model system, we used thiol-ene step growth polymerization with thiol-modified gelatin (GelSH) and PEG-norbornene linker; the technology was also applicable to other photo-crosslinkable chemistries, including gelatin methacryloyl (GelMA) and gelatin norbornene (GelNB) with PEG-thiol linker. The on-chip patterning strategy generated 3D cultures that were self-standing and that could be combined using serial photomasks. The method consistently generated features as small as 100 μm in diameter, and shared, non-linear boundaries between cultures were readily achieved. The modularity of the platform meant that designs were interchangeable in the same microfluidic housing, without requiring new master fabrication. As a proof-of-principle, a fragile cell type, primary human T cells, were patterned in varied geometries. Cells were patterned with high regional specificity and viability remained high. We expect that this technology will enable researchers to organize 3D cultures into geometries that were previously difficult to obtain, granting access to biomimetic tissue organizations and 3D-cultured microarray formats.