Nanoscale surface topography exerts profound effects on stem cell behaviour. Detailed knowledge of these effects is of key relevance to the development of advanced materials for tissue engineering. In order to gain such knowledge, high-throughput platforms for characterising cell-surface interactions are required. One suitable platform technology involves the use of lateral gradients where a continuous variation in a chemical or physical surface parameter of choice is investigated. Here, we fabricate porous silicon (pSi) gradients displaying pore sizes ranging from approximately 2 μm down to a few nm in diameter, and changing in solid surface fraction from 15% to 95% along the 12 mm length of the gradient. Cell attachment and spreading of primary rat bone marrow-derived mesenchymal stem cells (rBMSCs), human BMSCs (hBMSCs) and primary human adipose-derived mesenchymal stem cells (hASCs) were studied on these gradients. For all three MSC types, spreading was suppressed on the large pore size region of the gradient and spreading increased towards the other end of the gradient. MSC attachment density, on the other hand, was pore size- and cell type-dependent. In contrast to hASCs, the attachment density of rBMSCs and hBMSCs was low on the pSi gradient compared with flat Si. For hASCs, we observed a peak attachment density on the pore size region of 329 ±112 nm. F-actin staining showed clearly that cytoskeleton development was related to both cell spreading and cell type. The result hence showed that the pore size gradient format allows the screening of suitable surface topography parameters for the control of MSC behaviour. We also observed that MSCs derived from different sources respond differently to pore size at the nanoscale, suggesting that intrinsic differences between MSCs affect cell-surface interactions and justifying the need for high-throughput platforms, such as the one developed here.