© Springer International Publishing Switzerland 2015. Diffusion magnetic resonance imaging (dMRI) provides a non-invasive tool to explore biological tissues, including brain with its highly organised hierarchical fibrous structures. An MR phantom is a test object with known size and material for the calibration of MR scanners and the validation of image processing algorithms. Despite extensive research on the development of brain-mimicking phantoms, there are significant problems with using the existing phantoms for dMRI. This chapter is designed to lead the reader through the development of brain-mimetic phantoms for application in dMRI. Our starting point is a brief introduction to the dMRI technique and phantoms previously developed to mimic brain tissues. The second section focuses on the preparation and characterization of novel physical phantoms composed of co-electrospun hollow microfibres. Finally, the evaluation of the developed co-electrospun phantoms is presented in the third section.