The bone marrow (BM) is a complex microenvironment in which hematopoietic stem and progenitor cells (HSPCs) interact with multiple cell types that regulate their quiescence, growth, and differentiation. These cells constitute local niches where HSPCs are confined and subjected to specific set of physical and biochemical cues. Endothelial cells forming the walls of blood capillaries have been shown to establish a vascular niche, whereas osteoblasts lying along the bone matrix organize the endosteal niche with distinct and specific impact on HSPC fate. The observation of the interaction of HSPCs with niche cells, and the investigation of its impact on HSPCs behavior in vivo is hindered by the opacity of the bone matrix. Therefore, various experimental strategies have been devised to reconstitute in vitro the interaction of HSPCs with distinct sets of BM-derived cells. In this chapter, we present a method to manufacture a pseudo BM-on-a-chip with separated compartments mimicking the vascular and the endosteal niches. Such a configuration with connected but distant compartments allowed the investigation of the specific contribution of each niche to the regulation of HSPC behavior. We describe the microfabrication of the chip with a maskless photolithography method that allows the iterative improvement of the geometric design of the chip in order to optimize the adaptation of the multicellular architecture to the specific aim of the study. We also describe the loading and culture of the various cell types in each compartment.