Optimal function of Hox transcription factors may require Meis and Pbx cofactors. Here we test the in vivo Meis-dependence of two zebrafish paralog group-1 (PG1) Hox proteins. Misexpression of Hoxb1a induces ectopic gene expression throughout the anterior nervous system, while Hoxb1b induces ectopic expression primarily in hindbrain rhombomere 2. These activities are drastically reduced when endogenous Meis function is disrupted, demonstrating that both proteins are Meis-dependent. Upon addition of Meis3, Hoxb1b mimics the more severe Hoxb1a phenotype, indicating that Hoxb1b requires higher Meis levels than Hoxb1a. Using chimeric proteins we map this difference to the N-terminus, which contains the transcription activation domain. Lastly, we demonstrate strong genetic interactions between meis and PG1 hox genes, as well as between meis and pbx genes, in vivo. Our results are consistent with PG1 hox genes requiring pbx and meis function in vivo and reveal that different Hox proteins have distinct Meis requirements.