Surface micro- and nanotopographies have shown potential in inducing osteogenic differentiation. Titanium nanopillars can enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). While the exact mechanisms by which osteogenic differentiation is induced remain unclear, mechanotransduction is thought to play an important role. This study investigated the effect of titanium nanopillars (bTi) on integrins, involved in the first phase of mechanotransduction, during the adhesion of human mesenchymal stromal cells (hMSCs). Titanium nanopillars were produced by inductively coupled plasma reactive ion etching (ICP RIE). The resultant nanopillars did not hinder cell adhesion but resulted in distinct cell morphologies, namely a predominant polygonal shape on the planar titanium control (fTi) and a stellate shape on bTi. A slower adaptation of cells on bTi relative to fTi was suggested by delayed cell spreading and enhanced filopodia development. RNA was isolated based on an optimised protocol developed in this study. Gene expression analysis revealed that all 10 integrin subunits involved in osteogenic differentiation were expressed by cells on both fTi and bTi during the 48 hours of culture. ITGA1 and ITGA4 were upregulated on bTi relative to fTi after 24 hours of culture. The upregulation of ITGA4 suggested increased migration of cells on bTi. Interestingly, ITGA2, ITGAV, and ITGB3 showed a peak at 24 hours on bTi. Immunocytochemical analysis was performed based on the protocol established in this study. The preliminary immunocytochemical analyses suggested fibrillar adhesions including integrin subunit α5 on fTi and both α2 and α5 on bTi. Further investigation of the mechanotransduction pathway may elucidate any correlation between integrin expression, actin reorganisation, and osteogenic differentiation of hMSCs cultured on such nanopillars and provide valuable insights for finding earlier osteogenic markers to be used for the evaluation of osteogenic nanotopographies.