In this work, a branched peptide amphiphile (B-PA) presenting RGD and IKVAV motifs was fabricated by solid-phase peptide synthesis and self-assembled into a nanofiber hydrogel in which rabbit bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured for seven days. Specifically, 1 wt% B-PA was self-assembled into a nanofiber hydrogel with the addition of culture medium and observed using transmission electron microscopy. The B-PA with a molecular weight of 2191.72 and a purity >95% self-assembled into nanofibers with diameters from 6 to 8 nm and lengths ranging from hundreds of nanometers to several micrometers. BMSCs were acquired from rabbits using differential adherence methods and identified by flow cytometry for cell phenotype. The cells were stained with calcein acetoxymethyl ester/propidium iodide to assess cell viability, CCK-8 to assess cell cytotoxicity and proliferation, and Hochest 33342 to assess cell adhesion. They were also immunofluorescently labeled with microtubule-associated protein-2 (MAP-2), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP) to assess cell transdifferentiation. The B-PA hydrogel provided platform upon which the CD29+/44+ cells adhered and proliferated, and it induced the transdifferentiation of cells into neural cells expressing the markers MAP-2, NF, and GFAP. The hydrogel exhibited good cytocompatibility and multiple functions, and may therefore serve as a scaffold for neural tissue engineering.