We developed the microwell arrays to control cell fate. To study the photothermal therapy, the multicellular tumor spheroids were grown inside the microwell arrays. Various types of cancer cells were cultured inside the microwell arrays for 2 days with nanoparticles. The multicellular tumor spheroid size was reduced. Also, live/dead cell viability assays demonstrated the nanoparticle-mediated photothermal therapy. In addition, to generate homogeneous neurosphere, neural stem cells (NSCs) were cultured in the microwell arrays for 3 days. The homogeneous neurospheres were replated into extracellular matrix (ECM)-treated two-dimensional (2D) substrate and were subsequently encapsulated by photocrosslinkable hydrogels in a three-dimensional (3D) manner. We analyzed the effect of microwell sizes on neuronal differentiation. Therefore, these microwell arrays could be powerful tools for studying stem cell differentiation and photothermal therapy applications.