Endogenous damage-associated molecular pattern (DAMP) molecules are released from cells during traumatic injury, allowing them to interact with pattern recognition receptors such as the toll-like receptors (TLRs) on other cells and subsequently, to stimulate inflammatory signaling. TLR4, in particular, plays a key role in systemic and remote organ responses to hemorrhagic shock (HS) and peripheral tissue injury in the form of bilateral femur fracture. TLR4 chimeric mice were generated to investigate the cell lineage in which functional TLR4 is needed to initiate the injury response to trauma. Chimeric mice were generated by adoptive bone marrow (BM) transfer, whereby donor marrow was given to an irradiated host using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuJ) and TLR4 mutant (Mu; C3H/HeJ) mice. After a period of engraftment, chimeric mice were then subjected to HS or bilateral femur fracture. Control groups, including TLR4-WT mice receiving WT BM and TLR4-Mu mice receiving Mu BM, responded to injury in a similar pattern to unaltered HeOuJ and HeJ mice, and protection was afforded to those mice lacking functional TLR4. In contrast, TLR4-WT mice receiving Mu BM and TLR4-Mu mice receiving WT BM demonstrated intermediate inflammatory and cellular damage profiles. These data demonstrate that functional TLR4 is required in BM-derived cells and parenchymal cells for an optimal inflammatory response to trauma.