We present the synthesis of hydrogel microbeads based on telechelic poly(2-oxazoline) (POx) crosslinkers and the methacrylate monomers (HEMA, METAC, SPMA) by inverse emulsion polymerization. While in batch experiments only irregular and ill-defined beads were obtained, the preparation in a microfluidic (MF) device resulted in highly defined hydrogel microbeads. Variation of the MF parameters allowed to control the microbead diameter from 50 to 500 μm. Microbead elasticity could be tuned from 2 to 20 kPa by the POx:monomer composition, the POx chain length, net charge of the hydrogel introduced via the monomer as well as by the organic content of the aqueous phase. The proliferations of human mesenchymal stem cells (hMSCs) on the microbeads were studied. While neutral, hydrophilic POx-PHEMA beads were bioinert, excessive colonization of hMSCs on charged POx-PMETAC and POx-PSPMA was observed. The number of proliferated cells scaled roughly linear with the METAC or SPMA comonomer content. Additional collagen I coating further improved the stem cell proliferation. Finally, a first POx-based system for the preparation of biodegradable hydrogel microcarriers is described and evaluated for stem cell culturing.