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The ability to control the spatiotemporal microenvironment of cultured cells, such as cells in soluble gradients of signaling molecules, is critical for understanding the quantitative relationship between physicochemical signals and the induced biological responses. Existing methods for generating biomolecule gradients either offer little control, result in poor reproducibility, and/or place cells in potentially confounding or adverse conditions. Here the authors report the generation of soluble gradients by a system of microfluidic “jets” that inject picoliter amounts of fluid into an open pool with negligible exposure of the surface to flow. A stable, reproducible gradient can be formed in minutes.