Helix-loop-helix (HLH) transcription factors are key regulators of neurogenesis, myogenesis and osteogenesis. Here the relative contributions of multiple classes of HLH factors to the expression of bone related genes during osteoblast maturation were compared. We examined the expression of a panel of HLH proteins (e.g., Twist1/2, USF1/2, c-Myc, Id1 approximately 4, E12/47, Stra13) and one Zn finger protein (Snail which recognizes a subset of E-boxes), during osteoblast differentiation and their functional contributions to bone phenotypic gene regulation. While expression of Twist1, Stra13, E12/47 and Snail transcripts remains relatively constant, expression of Twist2 as well as the inhibitory factors Id1, Id2, Id3, and Id4 decreases and USF1 is up-regulated during osteoblastic differentiation of MC3T3 cells. Forced expression of selected HLH transcription factors shows that Myc, Snail and USF factors increase expression of the bone markers osteocalcin (OC) and/or alkaline phosphatase (AP), while E12/47, Twist and Id factors decrease their expression. None of these factors affect Runx2 gene expression. Interestingly, Snail enhances expression of osteoblast markers, while Twist1 and Twist2 factors are cross-regulated and inhibit bone specific gene expression and other HLH proteins (e.g., Id) indirectly. Thus, our data suggest that the integrated activities of negative and positive E-box related regulatory factors control osteoblast differentiation.