Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more eficacious treatments. Here, we introduce a novel micro-/ nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (> 10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ~4fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living- cell-intererogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.