Skeletal stem cells (SSCs) are a sub-population of bone marrow (BM) stromal cells with multipotent differentiation potential. SSCs are responsible for the unique regeneration capacity inherent to bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge hampering their clinical translation remains the isolation of homogeneous SSC populations with consistent regeneration and differentiation capacities. Factors limiting the efficiency of existing sorting approaches include the scarcity of SSCs in BM, estimated at fewer than 1 in 10,000 nucleated cells, the complexity of BM tissue and, most significantly, the absence of a specific marker that is unique to the SSC. Microfluidics offers the potential to characterise and sort cells marker-free, based on intrinsic biophysical properties. These include, but are not limited to, cell size, shape, stiff­ness, and dielectric properties. The work presented herein aimed to provide a comprehen­sive characterisation of the biophysical fingerprint of SSCs and to build on this new understanding to develop new tools to isolate SSCs, label-free, with significant physiological and therapeutic implications. In real-time deformability cytometry (RT-DC), cells are deformed by shear and normal stresses as they flow through a narrow constriction at high speed, providing the capability to screen cell mechanical properties at high-throughput. Here, RT -DC was used to relate the mechano-phenotype of expanded SSCs with other cells in BM. Critically, SSCs were found to be significantly stiffer than white blood cells, which are abundant in human BM. Microfluidic impedance cytometry was coupled to fluorescence optical detection to provide accurate characterisation of the dielectric properties and cell size of SSCs within heterogeneous primary human BM samples. The membrane capacitance of SSCs was found to be indistinct from other cells in BM. Conversely, their average size in suspension, at 9 micrometres, was within the largest BM cell fraction. Centred on these findings, label-free sorting devices were designed based on the prin­ciple of deterministic lateral displacement (DLD). DLD uses arrays of micropillars in a chan­nel to sort cells based on their diameter, at throughputs of thousands per second. Cell deformation, induced by shear and contact with the pillars, can change the effective cell size and affect sorting efficiency. This was demonstrated using two human cells lines of different size and stiffness, and by size fractionation of expanded SSCs. Crucially, SSCs sorted by DLD remained viable and retained their capacity to form clonogenic cultures. Overall, this work provided a detailed characterisation of relevant biophysical proper­ties of SSCs and paved the way towards the design of a novel label-free sorting approach, potentially based on DLD, to provide purified SSC populations from BM with impactful use in fundamental stem cell research and the clinic.