Aim Cell culture system with glass-like rigid substrates is currently used to detect cell biological behaviors. However, the impact of the stiffness of tissue-like soft substrates on stem cell morphology and differentiation is rarely considered in cell-based cardiac repair. This work aimed to access the effects of myocardium-like soft culture substrates on the specification of CD34⁺ as well as CD34－ subsets along endothelial lineage in comparison with conventional glass-like rigid substrates. Methods Elastic modulus (E, a material property that describes the stiffness or elasticity) of normal myocardium was measured using atomic force microscopy (elastic modulus≈15 kPa). Myocardium-like soft culture substrates were prepared using polyacrylamide gel with a similar stiffness to normal myocardium. Meanwhile, cell culture system with glass-like rigid substrates (elastic modulus >1 GPa) was used as the control. Mouse bone marrow-derived CD34⁺ and CD34－ cells were collected by density gradient centrifugation and magnetic activated cell sorting (MACS). The isolated cells were cultured both on myocardium-like soft matrix and glass rigid substrate (E>1 GPa). At day 7 of culture, the surface markers of endothelial lineage, cell morphology, and cytoskeleton were observed by laser scanning confocal microscopy. Results Regardless of the substrate stiffness, mouse bone marrow-derived CD34⁺ cell subsets exhibited higher percentage of double-positive cells for dil-labelled acetylated-low density lipoprotein (Dil-ac-LDL) uptake and FITC-labelled ulex europaeus agglutinin I lectin (FITC-UEA-1) binding, and higher expression of endothelial lineage markers, CD31, vWF, Flk-1, and VE-cadherin than CD34－ subsets. Moreover, in terms of the difference in cell specification efficacy between CD34⁺ subsets and CD34－ subsets, myocardium-like soft substrate showed a more potent induction capacity than conventional glass-like rigid substrate. It might partially result from more stressful F-actin fibers and more abundant focal adhesions of CD34⁺ cell subsets on myocardium-like soft substrates. Conclusions Myocardium-like soft substrate was capable of inducing potently cell specification of CD34⁺ subsets compared with glass-like rigid substrate. It speculated that tissue-like soft substrate might be a more optimal culture system for detecting the specification of stem cells in vitro.