The development of transitional interfacial zones between adjacent tissues remains a significant challenge for developing tissue engineering and regenerative medicine strategies. Using osteogenic differentiation as a model, we describe a novel approach to spatially regulate expression and secretion of the bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrate control of gene expression, and of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we design laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of BMP-2. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Patterned differentiation experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. In summary, by combining inducible gene expression with laminar flow technologies, this study provided an innovative way to engineer tissue interfaces.