High Mobility Group Box 1 protein in its fully-reduced state (FR-HMGB1) binds to CXC Ligand 12 (CXCL12) and signals through CXC Receptor 4 (CXCR4) to transition stem and progenitor cells from the quiescent G0state to GAlert. On exposure to tissue activating factors, cells in GAlertreadily enter G1to effect tissue repair. However, conversion of FR-HMGB1 to the disulfide form may result in deleterious inflammation through signaling via Toll-Like Receptors 2 (TLR-2) and 4 (TLR-4), and the Receptor for Advanced Glycation End products (RAGE). Using peptide arrays, biolayer interferometry and nuclear magnetic resonance, we mapped the residues and motifs of HMGB1 that interact with CXCL12, TLR-2, TLR-4, and RAGE. Identification of repeating sequences across the Box A and Box B domains that interact with CXCL12 enabled us to design a construct comprising two HMG B Boxes in tandem (dBB12L), which promotes tissue repair as effectively as wild type FR-HMGB1 but is unable to signal through TLR-2, TLR-4 or RAGE.