Human osteoclasts were differentiated using receptor activator of NFκB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) from colony forming unit-granulocyte macrophage (CFU-GM) precursors of the myeloid lineage grown from umbilical cord blood. Gene expression profiling using quantitative polymerase chain reaction (Q-PCR) showed more than 1,000-fold induction of chemokine MCP-1 within 24 h of RANKL treatment. MCP-1 mRNA content exceeds that of other assayed chemokines (CCL1, 3, 4, and 5) at all time points up to day 14 of treatment. MCP-1 induction preceded peak induction of calcium signaling activator calmodulin 1 (CALM1) and transcription factors JUN and FOS, which were at 3 days. Key osteoclast related transcription factors NFATc1 and NFATc2 showed peak induction at 7 days, while marker genes for osteoclast function cathepsin K and tartrate resistance acid phosphatase (TRAP) were maximally induced at 14 days, corresponding with mature osteoclast function. To test whether the early and substantial peak in MCP-1 expression is part of human osteoclast differentiation events, a dominant negative inhibitor of MCP-1 (7ND) was added simultaneously with RANKL and M-CSF, resulting in blockade of CALM1, JUN and NFATc2 induction and strong inhibition of human osteoclast differentiation. These data show that a cascade of gene expression leading to osteoclast differentiation depends on intact early MCP-1 induction and signaling in human osteoclasts. J. Cell. Biochem. 115: 303–312, 2014. © 2013 Wiley Periodicals, Inc.