Jane was probably born at Wulfhall, Wiltshire, although West Bower Manor has also been suggested, the daughter of Sir John Seymour and Margery Wentworth. Her birth date was not recorded, but it is generally estimated as occurring in or around 1508. Through her maternal grandfather, she was a descendant of King Edward III's son Lionel of Antwerp, 1st Duke of Clarence. Because of this, she and King Henry VIII were fifth cousins.

Henry VIII was betrothed to Jane on 20 May 1536, just one day after Anne Boleyn's execution. The couple were married at the Palace of Whitehall, Whitehall, London, in the Queen's closet by Bishop Gardiner on 30 May 1536. As a wedding gift the King made her a grant of 104 manors in four counties as well as a number of forests and hunting chases for her jointure, the income to support her during their marriage. She was publicly proclaimed queen on 4 June 1536. Jane's well-publicised sympathy for the late Queen Catherine and her daughter Mary showed her to be compassionate and made her a popular figure with the common people and most of the courtiers. She was never crowned because of plague in London, where the coronation was to take place. Henry may have been reluctant to have Jane crowned before she had fulfilled her duty as a queen consort by bearing him a son and a male heir. As queen, Jane Seymour was said to be strict and formal. Jane would form a close relationship with her stepdaughter, Mary. The lavish entertainments, gaiety, and extravagance of the queen's household, which had reached its peak during the time of Anne Boleyn, was replaced by a strict enforcement of decorum. For example, she banned the French fashions that Anne Boleyn had introduced. Politically, Seymour appears to have been conservative. Her only reported involvement in national affairs, in 1536, was when she asked for pardons for participants in the Pilgrimage of Grace. Henry is said to have rejected this, reminding her of the fate her predecessor met with when she "meddled in his affairs". Her motto as a queen was "Bound to obey and serve."  Jane put forth much effort to restore Mary to court and to the royal succession, behind any children that Jane might have with Henry. Jane brought up the issue of Mary's restoration both before and after she became queen. While Jane was unable to restore Mary to the line of succession, she was able to reconcile her with Henry. Eustace Chapuys wrote to Charles V of Jane's compassion and efforts on behalf of Mary's return to favour. A letter from Mary to Jane shows that Mary was grateful to Jane. While it was Jane who first pushed for the restoration, Mary and Elizabeth were not reinstated to the succession until Henry's sixth wife, Catherine Parr, convinced him to do so.  In January 1537, Jane became pregnant. During her pregnancy, she developed a craving for quail, which Henry ordered for her from Calais and Flanders. During the summer, she took no public engagements and led a relatively quiet life, being attended by the royal physicians and the best midwives in the kingdom. She went into confinement in September 1537 and gave birth to the coveted male heir, the future King Edward VI, at two o'clock in the morning on 12 October 1537 at Hampton Court Palace. Edward was christened on 15 October 1537, without his mother in attendance, as was the custom. He was the only legitimate son of Henry VIII to survive infancy. Both of the King's daughters, Mary and Elizabeth, were present and carried the infant's train during the ceremony.

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