Surfaces modified with thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) support mild and efficient harvesting of anchorage-dependent cells. To enable cellular detachment, however, the surfaces must exhibit a narrow range of PNIPAAm thicknesses. In this work, this limitation is circumvented by introducing nanopatterns to grafted PNIPAAm brushes, eliminating the critical thickness requirement for cell-culturing applications. Nanopatterned PNIPAAm surfaces are prepared using a combination of interferometric lithography (IL) and surface-initiated polymerization. Above the lower critical solution temperature (LCST) of PNIPAAm (∼32 °C), these surfaces support the attachment and proliferation of mammalian cells (e.g., fibroblasts and endothelial cells). Below the LCST of PNIPAAm, cells readily detach from the nanopatterned PNIPAAm surfaces without influence from the period of nanopatterns, which vary between 157 ± 9 nm to 1021 ± 17 nm. Cells selectively attach and proliferate on PNIPAAm nanopatterns as compared to thick unpatterned PNIPAAm, which is further exploited to spatially direct cellular growth to generate cellular micropatterns. Nanopatterned PNIPAAm surfaces provide a unique solution to the critical thickness issue for cell harvesting and facilitate spatial control of cellular growth on surfaces.