Upland cotton (Gossypium hirsutum) is one of the most recalcitrant species for in vitro plant regeneration through somatic embryogenesis. Callus from only a few cultivars can produce embryogenic callus (EC), but the mechanism is not well elucidated. Here we screened a cultivar, CRI24, with high efficiency of EC produce. The expression of genes relevant to EC production was analyzed between the materials easy to or difficult to produce EC. Quantitative PCR showed that CRI24, which had a 100% EC differentiation rate, had the highest expression of the genes GhLEC1, GhLEC2, and GhFUS3. Three other cultivars, CRI12, CRI41, and Lu28 that formed few ECs expressed these genes only at low levels. Each of the genes involved in auxin transport (GhPIN7) and signaling (GhSHY2) was most highly expressed in CRI24, with low levels in the other three cultivars. WUSCHEL (WUS) is a homeodomain transcription factor that promotes the vegetative-to-embryogenic transition. We thus obtained the calli that ectopically expressed Arabidopsis thaliana Wus (AtWus) in G. hirsutum cultivar CRI12, with a consequent increase of 47.75% in EC differentiation rate compared with 0.61% for the control. Ectopic expression of AtWus in CRI12 resulted in upregulation of GhPIN7, GhSHY2, GhLEC1, GhLEC2, and GhFUS3. AtWus may therefore increase the differentiation potential of cotton callus by triggering the auxin transport and signaling pathways.