This paper describes three-dimensional (3D) tissue shape control of mouse neural stem cell (mNSC) micro tissues by using closed agarose microchambers for effective differentiation induction of neurons in vitro. Our agarose microchambers, made by micromolding, can be sealed with an agarose sheet to form the mNSC tissues along the shape of microchambers. We constructed lane-shaped mNSC tissues with different width (approximate to 60-210m) and thickness (approximate to 25-95m) dimensions and induced differentiation to neurons with differentiation medium. We found that in thick tissues (thickness: >60m), distribution of differentiated neurons was not uniform, whereas in thin tissues (thickness: approximate to 30m), differentiated neurons were uniformly distributed with high differentiation efficiency. Our system to construct in vitro 3D neural tissues having uniformly distributed neurons at high differentiation ratio, could become an effective tool for drug screening using 3D neural tissues and 3D mNSC tissues under differentiation induction.