Microtubule organization in living cells is determined by spatial control of microtubule nucleation, their dynamic properties, and transport by molecular motors. Here, we establish a new micropattern-guided method for controlling local microtubule nucleation by spatially confined immobilization of a microtubule polymerase and show that these nucleated microtubules can be transported and organized in space by motor proteins. This assay provides a new platform for deciphering the principles underlying mesoscale microtubule organization. © 2013 American Chemical Society.