Controlling the spatial arrangement of biomaterials, including living cells, with high resolution (±5 μm) provides the foundation for fabricating complex biologic systems for studies ranging from the fundamentals of cell-cell and cell-matrix interactions to applications in tissue engineering. However, the level of spatial control required cannot be obtained through conventional cell processing techniques (e.g., pipetting of or even ink-jetting cells), as they lack the precision, reproducibility, and speed required for the rapid fabrication of idealized engineered constructs. Laser direct-write approaches (e.g., matrix-assisted pulsed-laser evaporation direct-write [MAPLE DW]), previously employed for the rapid prototyping of electronics, have shown reliable patterning of biomaterials with a spatial resolution of ±5 μm. Moreover, recent advances allow the rapid, precise deposition of viable mammalian cells and pluripotent stem cells on well-defined substrates, enabling the laser direct-writing platform to advance manipulation of the in vitro cellular microenvironment (e.g., the stem cell niche). Herein, we review the mechanisms and recent advances demonstrating the versatility and biofabrication potential of one particular laser-based technique, MAPLE DW.