Ultrashort amphiphilic peptides, comprised of concise sequences of seven to four amino acids, display distinctive self-assembling behavior in physiological conditions, forming nanofiber-rich hydrogels with remarkably high water content (>99% w/w). The hydrogels are comprised of nanofibers and three-dimensional (3D) matrices mirroring the structure of the extracellular matrix. These hydrogels exhibit biocompatibility, creating a nurturing microenvironment that supports the viability, proliferation, and differentiation of stem cells.With the aim of enhancing the peptide scaffolds through integration with human platelet lysate while preserving the self-assembly process, this study explores the synergistic potential of ultrashort peptides and platelet lysates in guiding stem cell behavior within the hydrogel matrix. In this investigation, a gelation test was performed to determine the minimum gelation concentration of the developed peptide. Additionally, we assessed cell viability, proliferation, as well as cell-cell and cell-matrix interactions. Through the strategic substitution of specific amino acids in the peptide sequence, we achieved the lowest reported minimum gelation concentration for self-assembling peptides, thereby expanding the range of their potential applications.By substituting specific amino acids in the peptide sequence, we successfully developed a peptide sequence that can form a nanofibrous hydrogel with the lowest documented minimum gelation concentration for self-assembling peptides, consequently broadening the scope of their potential applications. The peptide hydrogel formed a highly porous network of nanofibers with tunable mechanical properties. The 3D peptide culture supported the viability and growth of various cell types, including mesenchymal stromal cells. Additionally, it recapitulated cell-cell and cell-matrix interaction. Notably, the developed 3D culture system was able to support the growth of cells using media without serum supplementation. This attribute allows for the provision of a xeno-free 3D culture system that could be employed for advanced biomedical applications.This innovative framework shows potential in transforming the landscape of stem cell therapies by providing customized and dynamic settings. The integration of ultrashort peptides and platelet lysates in hydrogel design represents a significant stride toward advancing the field of stem cell-based therapies.